RU2610664C2 - Method of controlling weeds using plants aad-1 and presowing and / or pre-emergence treatment with herbicides - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biochemistry and more particularly to a method of controlling weeds, comprising seeding a certain area and treating with aryloxyalkanoate herbicide on the said area 30 days before sowing of seeds in the said area, where the said seeds contain aryloxyalkanoate dioxygenase AAD-1 protein encoded by the sequence of SEQ ID NO: 29.

EFFECT: invention can effectively control weeds in a certain area containing crop plants.

12 cl, 7 dwg, 33 tbl, 13 ex

Description

BACKGROUND

The aad-1 gene (originally known from Sphingobium herbicidovorans) encodes an aryloxyalkanoate dioxygenase (AAD-1) protein. This feature confers herbicide resistance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate (commonly called “fop” herbicides such as quizalofop) and can be used as a selectable marker in plant transformation and in breeding nurseries. The aad-1 gene, as such, in connection with the resistance of plants to herbicides was first disclosed in WO 2005/107437 (also see US 2009-0093366).

The expression of heterologous or foreign genes in plants is influenced by where the foreign gene is inserted in the chromosome. This may be due, for example, to the structure of chromatin (e.g., heterochromatin) or the proximity of transcriptional regulatory elements (e.g. enhancers) to the site of integration (Weising et al., Ann. Rev. Genet. 22: 421-477, 1988). The same gene in the same type of transgenic plant (or other organism) can exhibit wide variability of expression level at different events. There may also be differences in spatial or temporal patterns of expression. For example, differences in the relative expression of the transgene in different plant tissues may not correspond to the patterns expected based on the elements of transcriptional regulation present in the introduced gene construct.

Thus, a large number of events are often created and screened to identify an event that results in the expression of the introduced gene of interest at a level sufficient for a given purpose. For commercial purposes, usually from several hundred to several thousand different events are carried out and such events are screened for a single event that provides the desired levels and patterns of transgene expression. An event that provides the desired levels and / or patterns of transgene expression is applicable for introgression of a transgene into another genetic environment as a result of sexual outcrossing using conventional selection methods. In offspring from such crosses, the expression characteristics of the transgene of the original transformant are preserved. This technique is used to ensure reliable gene expression in some varieties that are well adapted to local growth conditions.

U.S. patent applications 20020120964 A1 and 20040009504 A1 relate to the genetic event PV-GHGT07 (1445) in cotton plants and compositions and methods for detecting it. WO 02/100163 relates to event MONI5985 in cotton plants and compositions and methods for its detection. WO 2004/011601 relates to event MON863 in maize plants and compositions and methods for detecting it. WO 2004/072235 relates to event MON88913 in cotton plants and compositions and methods for its detection.

WO 2006/098952 relates to event 3272 in corn. WO 2007/142840 relates to event MIR162 in corn.

US patent No. 7179965 relates to cotton in which the cry1F event and the cry1Ac event occurred. Corn AAD-1, in which a particular event occurred, disclosed in the present description, has not been previously described.

SUMMARY OF THE INVENTION

The present invention relates to pre-sowing and / or pre-emergence application of the herbicide in an area or field that is seeded with seeds in which the AAD-1 event occurs. In some preferred embodiments, the DAS-40278-9 event of maize occurs in the seed. In some preferred embodiments, the herbicide may be a composition containing the active ingredient 2,4-D. Such herbicides and formulations can also be used for presowing application. Additional herbicides such as glyphosate can be used in combination, including presowing use.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a map of plasmid pDAS1740.

Figure 2 shows the insert components for DAS-40278-9 (pDAS1740).

Figure 3 shows a restriction map and insert components for DAS-40278-9 (pDAS1740).

The figure 4 shows the amplicons, primers and the cloning technique in the case of the DNA insert and the border of DAS-40278-9.

Figure 5 illustrates the position of the primers with respect to the insert and the boundaries of DAS-40278-9.

Figure 6 illustrates the connection regions and insertion of DAS-40278-9.

Figure 7 is a cross-breeding diagram described in Example 7.

BRIEF DESCRIPTION OF SEQUENCES

The sequences of SEQ ID NO: 1-28 are the primers that are described in this publication.

SEQ ID NO: 29 represents the insert and flanking sequences for the event in question DAS-40278-9.

The sequences of SEQ ID NO: 30-33 are primers for flanking markers, which are described in example 4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes pre-sowing and / or pre-emergence application of the herbicide to an area or field, which is then seeded with seeds in which the AAD-1 event occurs. In some preferred embodiments, the DAS-40278-9 event of maize occurs in the seed. In some preferred embodiments, the herbicide may be a composition containing the active ingredient 2,4-D. Such herbicides and formulations can also be used for presowing application. Additional herbicides such as glyphosate can be used in combination, including presowing use. The present invention is not limited to corn, but may include, for example, the use of cotton and / or soybeans containing the aad-1 gene.

The examples included herein relate in part to presowing and / or pre-emergence application of herbicides. Such applications are not limited to event "278". In addition, the applicability of the resistance provided by the AAD-1 genes can be explained taking into account the shortened period of time from application to new seeding. This provides plant growers with much greater flexibility in scheduling their crops relative to the moment of complete destruction of weeds (“burning”). Without the use of the present invention, waiting for 7-30 days or so after the destruction of the weeds before sowing can cause a significant loss of yield. Thus, the present invention provides advantages in this regard. See, for example, Example 13. Any intervals between sowing / applying herbicides and any concentration ranges / application rates of the herbicide (s) given as examples or proposed in the present description can be used according to the present invention.

Thus, the present invention relates to new methods of using herbicides. Such applications may include tank mixtures of more than one herbicide. Some preferred herbicides for use in the present invention include a phenoxy-auxin herbicide such as 2,4-D; 2,4-DB; MCPA; MCPB. Such applications can be combined with one or more additional herbicide resistance genes and a corresponding herbicide (e.g. glyphosate and / or glufosinate). One, two, three or more herbicides can be used in advantageous combinations, which may be obvious to a person skilled in the art, having the effect of the present invention. One or more of the proposed herbicides can be applied to the field / area before sowing according to the present invention. Such application can be carried out, for example, within 14 days before sowing. One or more of the proposed herbicides can also be applied during sowing and / or after sowing, but before germination. One or more of the proposed herbicides can also be applied to the soil (for weed control) or over weeds and / or transgenic plants according to the present invention. The three herbicides in question can be alternated or used in combination, for example, to control or prevent the emergence of weeds that can be resistant to one herbicide, but not to another. You can use different application times of the three types of herbicides in question in different ways that may be known in the art.

Thus, the present invention also relates to the pre-sowing application of the herbicide in an area or field, which is then seeded with seeds in which the AAD-1 event takes place. In some preferred embodiments, the DAS-40278-9 event of maize occurs in the seed. In some preferred embodiments, the herbicide may be a composition containing the active ingredient 2,4-D. Such herbicides and compositions can be used for presowing application. Additional herbicides, such as glyphosate, can be used in combination when presowing. In any of these options, for example, corn, cotton and soy can be used.

The aad-1 gene can be combined, for example, with characters encoding glyphosate resistance (e.g., resistant plant or bacterial EPSPS, GOX, GAT), glufosinate resistance (e.g. Pat, bar), resistance to acetolactate synthase inhibiting herbicide (ALS) (for example, imidazolinones [such as imazetapyr], sulfonylurea, triazolopyrimidinesulfonanilide, pyrimidinylthiobenzoates and other chemical compounds [Csr1, SurA and others]), resistance to bromoxynil (for example, Bxn), resistance to HPP 4 enzymes (enzyme inhibitors iphenylpyruvate dioxigenase), resistance to phytoindesaturase inhibitors (PDS), resistance to herbicides that inhibit photosystem II (e.g. psbA), resistance to herbicides that inhibit photosystem I, resistance to herbicides that inhibit protoporphininogen oxidase (1), PP (IX), PP (PPO) IX (PPO) phenylurea-based herbicide resistance (e.g., CYP76B1), dicamba-degrading enzymes (see, e.g., US 20030135879), and others that can be stacked individually or in many different combinations to allow for and effectively control weeds or prevent weed shift and / or resistance to any herbicide classes mentioned above.

With regard to additional herbicides, some additional preferred ALS inhibitors (also known as AHAS) include triazolopyrimidinesulfonanilides (such as cloransulam methyl, diclosulam, florosulam, flumetsulam, metosulam and pencoxulam), pyrimidinylthiobenzoates (such as pionibiribozybiburobenzoic. Some preferred HPPD inhibitors include mesotrione, isoxaflutol and sulcotrione. Some preferred PPO inhibitors include flumiclorac, flumioxazine, flufenpyr, piraflufen, flutiacet, butafenacil, carfentrazone, sulfentrazone and diphenyl ethers (such as acifluorfen, fomesafen, lactofen and oxyfluorfen).

AAD-1 genes for use in the present invention can also provide resistance to compounds that are converted to phenoxyacetate auxin herbicides (e.g., 2,4-DB, MCPB, etc.). The remainder of butyric acid present in the 2,4-DB herbicide is converted by β-oxidation to phytotoxic 2,4-dichlorophenoxyacetic acid. Similarly, MCPB is transformed through β-oxidation into phytotoxic MCPA. Butyric acid herbicides are not in themselves herbicidal. They turn into their corresponding acid as a result of β-oxidation in sensitive plants, and such an acetic acid form of the herbicide is phytotoxic. Plants incapable of rapid β-oxidation do not suffer from butyric acid herbicides. However, plants that are capable of rapid β-oxidation and can convert the butyric acid herbicide to the acetic acid form are subsequently protected by the present AAD-1.

The present invention covers the AAD-1 event in corn, designated DAS-40278-9, having seeds deposited in the American Type Culture Collection (ATCC) with accession number PTA-10244, and offspring derived from them. Other aspects include offspring plants, seeds and grains, or regenerated parts of plants and seeds and offspring of maize in the event of DAS-40278-9, as well as feed and food derived therefrom. The present invention also encompasses portions of maize plants where DAS-40278-9 occurs, which includes, but is not limited to pollen, ovule, flowers, shoots, roots and leaves and nuclei of vegetative cells, pollen cells and egg cells. In addition, maize plants are disclosed that are resistant to phenoxy-auxin and / or aryloxyalkanoate herbicides, to new genetic compositions in the case of DAS-40278-9 in maize and aspects of the agronomic efficacy of maize plants having the maize DAS-40278-9.

The present invention encompasses plant breeding methods and herbicide resistant plants, including the aad-1 transformation event in maize plants containing the polynucleotide sequence described herein embedded in a specific portion of the maize cell genome.

In some embodiments, the specified event / polynucleotide sequence may be “stacked” with other characteristics, including, for example, other herbicide resistance gene (s) and / or insect-suppressing proteins. Also in this publication, plants having a single event are described.

Additional traits can be stacked in the genome of the plant by crossing plants, re-transforming the transgenic plant having the DAS-40278-9 event of maize, or adding new traits as a result of targeted integration through homologous recombination.

Other options include excision of polynucleotide sequences in which the maize DAS-40278-9 event occurred, including, for example, the pat gene expression cassette. After excision of the polynucleotide sequence, the modified event can be redirected to a specific region of the chromosome where additional polynucleotide sequences are stacked with the DAS-40278-9 event of maize.

In one embodiment, the invention relates to a target site on a corn chromosome located on chromosome 2 at a position of about 20 cM between the SSR markers of UMC 1265 (see SEQ ID NO: 30 and SEQ ID NO: 31) and MMC0111 (see SEQ ID NO: 32 and SEQ ID NO: 33), about 20 cM on the 2008 DAS maize linkage map, with the target site containing a heterologous nucleic acid. In another embodiment, the invention relates to a target site in the corn chromosome containing the position determined in the sequence or sequence of SEQ ID NO: 29, and its residues, which are described in this publication, as will be understood by a person skilled in the art.

In one embodiment, the invention relates to a method for producing a transgenic maize plant comprising a heterologous nucleic acid insert on chromosome 2 at a position of about 20 cM between the SSR markers of UMC1265 (see SEQ ID NO: 30 and SEQ ID NO: 31) and MMC0111 (see SEQ ID NO: 32 and SEQ ID NO: 33) at a position of approximately 20 cM on the 2008 DAS corn clutch map. In yet another embodiment, the embedded heterologous nucleic acid is flanked from the 5'-side of the whole or part of the 5'-flanking sequence, which is defined in the present description with reference to the sequence of SEQ ID NO: 29, and flanked from the 3'-side of the whole or part of 3 ' the flanking sequence as defined herein with reference to the sequence of SEQ ID NO: 29.

In addition, assays are disclosed herein to detect the presence of a given event in a sample (e.g., corn grain). Assays can be based on the DNA sequence of a recombinant construct integrated into the maize genome and on genomic sequences flanking the insertion site. Kits and conditions applicable for analysis are also offered.

Also in the present description, the cloning and analysis of DNA sequences of the full insert of AAD-1 and its boundary regions (in transgenic maize lines) is disclosed. Such sequences are unique. Based on such insertion sequences and boundaries, event-specific primers were created. PCR analysis showed that such events can be identified by analyzing PCR amplicons generated using such event-specific primer sets. Thus, these and other related methods can be used to uniquely identify the lines of corn in which such an event takes place.

The present invention includes the use of crossing plants and herbicide resistant plants. The present invention includes new uses for transformation events in maize plants containing the aad-1 polynucleotide sequences described herein, which are embedded in a specific site in the maize cell genome. In some embodiments, said polynucleotide sequence may be “stacked” with other characters (such as another herbicide resistance gene (s), which are encoded, for example, by insect-suppressing proteins. In some embodiments, said polynucleotide sequences can be excised and then redirected to the target with additional polynucleotide sequences, however, the present invention includes plants in which there is a single event, which is described in this paragraph ublikatsii.

In addition, the present invention relates to assays to detect the presence of a given event in a sample. Aspects include methods for constructing and / or preparing any diagnostic nucleic acid molecules, exemplified or provided herein, in particular, molecules based entirely or partially on the flanking sequences in question.

More specifically, the present invention relates in part to the transgenic event DAS-40278-9 in maize (also known as pDAS 1740-278), to the use of plant lines in which such events occur, and the cloning and analysis of DNA sequences of such an insert and / or its border areas. Plant lines for use according to the present invention can be identified using the sequences disclosed and proposed in the present description. In some embodiments, the present invention relates to herbicide resistant maize lines and their identification. The present invention relates in part to detecting the presence of a given event in order to determine whether the offspring of sexual interbreeding has an event of interest. In addition, an event detection method is included, and such a method is applicable, for example, in order to ensure compliance with regulations requiring pre-sale permission and labeling of products obtained, for example, from recombinant cultivated plants. The presence of this event can be detected by any well-known method for detecting nucleic acid, such as polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. Event-specific PCR analysis is discussed, for example, by Windels et al. (Med. Fac. Landbouww, Univ. Gent 64 / 5b: 459462, 1999). The discussion relates to the identification of glyphosate-resistant event 40-3-2 in soy through PCR using a set of primers covering the connection between the insert and flanking DNA. More specifically, one primer contained the sequence from the insert, and the second primer contained the sequence of flanking DNA.

Maize was modified using the insertion of the aad-1 gene from Sphingobium herbicidovorans, which encodes an aryloxyalkanoate dioxygenase (AAD-1) protein. The attribute confers resistance to herbicides 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate (commonly called “fop” herbicides such as quizalofop) and can be used as a selectable marker in plant transformation and in breeding nurseries. Transformation of maize with a DNA fragment from plasmid pDAS1740 was carried out using crossbreeding, resulting in event DAS-40278-9.

Samples of genomic DNA extracted from twenty individual corn plants obtained from five generations, four plants per generation of the DAS-40278-9 event, were selected for the molecular characterization of the DAS-40278-9 event for AAD-1 corn. AAD-1 protein expression was tested using a set of strips for a quick AAD-1 specific test. For the subsequent molecular characterization, only plants were selected that were tested as positive for AAD-1 protein expression. Southern hybridization confirmed that the aad-1 gene is present in corn plants, which in the test were identified as positive for the expression of the AAD-1 protein, and the aad-1 gene was inserted as a single intact copy in such plants when hybridization with aad-1 gene probe.

The publication also describes the molecular characterization of the inserted DNA in the case of the DAS-40278-9 event for AAD-1 maize. The event was obtained by piercing cells by shaking them in a suspension of microneedles (Whiskers) using the Fsp I fragment of plasmid pDAS1740. Southern blot analysis was used to establish the integration pattern of the inserted DNA fragment and determine the number of insertions / copies of the aad-1 gene in event DAS-40278-9. Data was obtained to demonstrate the integration and integrity of the aad-1 transgene integrated into the maize genome. Characterization of integration of non-coding regions (intended for regulation of coding regions), such as promoters and terminators, regions of attachment to the RB7 Mar v3 and RB7 Mar v4 matrices was carried out, and the stability of the transgene insertion over generations was evaluated. The stability of the integrated DNA is shown over five separate generations of plants. In addition, the absence of the backbone sequence of the transforming plasmid, including the region of the ampicillin resistance gene (Ap ^r ) region, using probes covering almost the entire backbone region flanking the restriction sites (Fsp I) of the pDAS1740 plasmid, was shown. A detailed physical insertion map was obtained based on such Southern blot analyzes of the DAS-40278-9 event.

The levels of AAD-1 protein in maize tissues were determined. In addition, an analysis of the composition of corn fodder and grain was performed to examine the equivalence between the isogenic non-transformed corn line and the transgenic corn line DAS-40278-9 (non-sprayed, sprayed 2,4-D, sprayed with quizalophop and sprayed with 2,4-D and quizalopa ) The agronomic characteristics of the isogenic non-transformed maize line were also compared with DAS-40278-9 maize.

Field expression, nutrient composition, and agronomic testing of the non-transgenic control and hybrid maize line containing aryloxyalkanoate dioxygenase-1 (AAD-1) were performed in the same year at six locations in Iowa, Illinois (2 locations), Indiana, Nebraska and Ontario in Canada. Expression levels are summarized in the present description for AAD-1 protein in leaves, pollen, root, forage, whole plant and grain, the results of agronomic determinations and analysis of the composition of fodder and grain samples in control and AAD-1 DAS-40278-9 maize plants are presented.

The soluble extractable AAD-1 protein was measured using an enzyme-linked immunosorbent assay (ELISA) in leaves, pollen, root, forage, whole plant and corn grain. Good average expression values were observed in root and pollen tissues, as discussed in more detail herein. Expression values were similar for all spray treatments, as well as for plots sprayed and non-sprayed with 2,4-D herbicides and quisalophop.

Composition analysis, including food constituents, minerals, amino acids, fatty acids, vitamins, anti-nutritional substances and secondary metabolites, was carried out in order to examine the equivalence of AAD-1 DAS-40278-9 maize (with or without treatment with herbicides) to the control. All results of the analysis of the composition of samples AAD-1 DAS-40278-9 were as good or better (biologically and agronomically) than the results obtained on the basis of control lines and / or ordinary corn, as in the case of agronomic data collected from control plots and corn plots AAD-1 DAS-40278-9.

As mentioned in the section "Background", the introduction and integration of the transgene into the genome of plants includes some random events (therefore, the name "event" is used for this insertion, which is expressed). That is, in the case of many transformation techniques, such as transformation using Agrobacterium, the “gene gun” and WHISKERS, it is impossible to predict where the transgene will be inserted. Thus, the identification of the flanking genomic DNA of a plant on both sides of the insert may be important for identifying the plant in which such an insertion event occurred. For example, PCR primers can be designed that create a PCR amplicon spanning the region of the junction of the insert and the host genome. Such a PCR amplicon can be used to identify a unique or different type of insertion event.

Since “events” are initially random events, as part of this disclosure, at least 2500 seeds of the corn line in which the event takes place have been deposited and made available to the public without restriction (but covered by patent law) in the American crop type collection (ATCC), 10801 University Boulevard, Manassas, VA, 20110. Deposit received ATCC PTA-10244 (Hybrid Yellow Seed Maize (Zea Mays L.) seeds: DAS-40278-9; deposited on behalf of Dow AgroSciences LLC ; Date of receipt of seed / line (s) in ATTC: Jun 10 La 2009; viability confirmed August 17, 2009). The specified deposit has been made and will be maintained in accordance with and under the terms of the Budapest Treaty regarding the deposit of seeds for the purposes of the patent procedure. The deposit will be maintained without restriction at the ATCC depository, which is a publicly accessible depositary, for a period of 30 years, or five years after the most recent request, or during the effective term of the patent, of any duration, and will be replaced if it becomes unviable during this period.

Deposited seeds are part of the disclosed invention. It is clear that corn plants can be grown from such seeds, and such plants are part of the present invention. The present invention also relates to DNA sequences found in such corn plants that are useful for detecting such plants and their offspring. Detection methods and kits according to the present invention can be aimed at identifying any one, two or even all three of these events, depending on the ultimate goal of such a test.

Definitions and examples are given in the present description to help describe the present invention and instruct specialists in this field in relation to the practice of the invention. Unless otherwise indicated, the terms should be understood in accordance with their normal use by those skilled in the art. The nomenclature of bases for DNA used is that specified in 37 CFR § 1.822.

As used herein, the term “offspring” means the offspring of any generation obtained from a parent plant in which the corn event DAS-40278-9 AAD-1 occurs.

A transgenic “event” is obtained as a result of the transformation of plant cells with heterologous DNA, i.e. a nucleic acid construct that includes a transgene of interest, regenerating a plant population resulting from the insertion of a transgene into a plant genome and selecting a particular plant characterized by insertion at a specific position in the genome. The term “event” refers to the parent transformant and progeny of the transformant that contain heterologous DNA. The term “event” also refers to offspring resulting from sexual outcrossing between a transformant and another variety that contains genomic / transgenic DNA. Even after repeated crossbreeding with the parental form, the integrated transgene DNA and flanking genomic DNA (genomic / transgenic DNA) from the transformed parent are present in the progeny from the cross in the same position on the chromosome. The term “event” also refers to DNA from the original transformant and its progeny containing the integrated DNA and flanking genomic sequence immediately adjacent to the integrated DNA, which can be expected to be transmitted to the offspring that receives the integrated DNA, including the transgene of interest, in the result of sexual interbreeding of one parent line that contains embedded DNA (for example, the original transformant and offspring resulting from self-pollination), and a parent line that is not erzhit inserted DNA.

A “connection sequence” encompasses the point at which DNA embedded in the genome is linked to DNA from the native maize genome flanking the insertion point, and the identification or identification of one of such connection sequences in the plant’s genetic material is sufficient to diagnose the event. Also included are DNA sequences that span insertions in the events described in this publication and similar lengths of flanking DNA. Specific examples of such diagnostic sequences are provided herein; however, other sequences that overlap insertions or insertion and genomic sequences are also diagnostic and can be used according to the present invention.

The present disclosure includes the identification of such flanking sequences, connection and insertion sequences. The corresponding PCR primers and amplicons are included. Methods of PCR analysis using amplicons that overlap the integrated DNA and its boundaries can be used to identify or identify commercialized transgenic varieties or lines of corn derived from patented transgenic corn lines.

The complete sequences of each of these inserts together with parts of the corresponding flanking sequences are presented in the present description as SEQ ID NO: 29. The coordinates of the insert and flanking sequences for this event with respect to the sequence of SEQ ID NO: 29 (total 8557 base pairs) are listed below. A more detailed discussion is given, for example, in example 3.8. Presowing embodiments of the present invention include the use of AAD-1 protein encoded by residues 1874-6689 of the sequence SEQ ID NO: 29.

5 ’Flanking Insert 3 ’Flanking Residue Numbers (Seq: 29) 1-1873 1874-6689 6690-8557 Length (bp) 1873 bp 4816 bp 1868 bp

Such an insertion event and its additional components are shown further in figures 1 and 2. Such sequences (in particular flanking sequences) are unique. Based on such insertion sequences and boundaries, event-specific primers were created. PCR analysis showed that such maize lines can be identified in different maize genotypes using the analysis of PCR applicons created using such event-specific primer sets. Thus, these and other related methods can be used to uniquely identify such lines of corn. The sequences described herein are unique. For example, searches using BLAST in GENBANK databases did not reveal any significant homology between cloned boundary sequences and sequences in the database.

Detection methods are especially applicable in conjunction with plant breeding to determine which plants in the offspring have a given event after the parent plant having the event of interest is crossed with another line plant in order to try to give one or more additional traits to the offspring. Such methods of PCR analysis are useful for implementing maize breeding programs, as well as for quality control, especially in the case of commercial production of transgenic corn grains. Kits for PCR detection of such transgenic maize lines can also be obtained and used. The methods may also be useful for product registration and product management.

In addition, flanking maize / genomic sequences can be used to specifically identify the position of each insert in the genome. Such information can be used to obtain molecular marker systems specific to each event. Such systems can be used for accelerated selection and clutch data acquisition systems.

In addition, information on flanking sequences can be used to study and characterize the transgene integration process, characteristics of the integration site in the genome, sorting events, stability of transgenes and its flanking sequences, and gene expression (especially regarding gene silencing, gene methylation patterns, position effects, and potentially related to the expression of elements such as MAR [sites of attachment to the matrix] and the like).

The present invention includes seeds available from ATCC with a deposit number of PTA-10244. The present invention also relates to the use of a herbicide-resistant corn plant grown from a seed deposited in ATCC under accession number PTA-10244. In addition, the present invention relates to the use of parts of said plant, such as leaves, tissue samples, seeds obtained from said plant, pollen, and the like.

In addition, the present invention relates to the use of descendant plants and / or descendants of a plant grown from a deposited seed, preferably herbicide resistant corn plant, said plant having a genome containing a registered wild type / DNA genomic DNA compound sequence insert, which is described in this publication. As used herein, the term “corn” means Zea mays corn and includes all varieties that can be crossed.

In addition, the present invention relates to methods for crossbreeding using a plant according to the present invention as at least one of the parents. For example, the present invention relates to the use of a hybrid plant F ₁ having as one or both parents any of the plants described in the present description as an example. Also within the scope of the present invention is the use of seed obtained from such F ₁ hybrids of the present invention. The present invention relates to a method for producing an F1 hybrid seed by crossing an exemplary plant with another plant (e.g., an inbred parent) and collecting the resulting hybrid seed. The present invention relates to the use of an exemplary plant, which is either a female parent or a male parent. The characteristics of the resulting plants can be improved by careful selection of the parent plants.

The selection of a herbicide-resistant corn plant can be accomplished using the first sexual crossing of the first parent corn plant, which is a corn plant grown from the seed of any of the lines described in the present description, and the second parent corn plant, to obtain many descendant plants of the first generations; and then using the selection of a first-generation offspring plant that is herbicide resistant (or a plant in which at least one of the events of the present invention takes place); and self-pollination of a first-generation offspring plant to produce multiple second-generation offspring plants; and then using selection from descendant plants of the second generation of a plant that is resistant to the herbicide (or a plant in which at least one of the events of the present invention takes place). Such steps may further include crossbreeding the first-generation offspring plant or the second-generation offspring plant with a second parent corn plant or third parent corn plant. You can then sow a grain crop containing corn seeds according to the present invention or its offspring.

It should also be understood that two different transgenic plants can also be crossed to produce offspring that contain two added exogenous genes, which are split independently. Self-pollination of the corresponding offspring can produce plants that are homozygous for both added exogenous genes. It also provides for crossbreeding with the parent plant and outcrossing with a non-transgenic plant, as well as vegetative propagation. Other methods commonly used to obtain different traits and cultures are known in the art. The cross-breeding selection was used to transfer genes of a simple and highly inherited trait into the desired homozygous cultivar or inbred line, which is a recurrent parent. The source of the trait to be transferred is called the donor parent. It is expected that the resulting plant possesses the properties of a recurrent parent (for example, a cultivated plant variety) and the required trait transferred from the donor parent. After the initial crossbreeding, individuals possessing the phenotype of the donor parent are selected and re-crossed (crossbreeding is carried out) with the recurrent parent. It is assumed that the resulting parent has the properties of a recurrent parent (for example, a cultivated plant variety) and the required trait transferred from the donor parent.

The DNA molecules disclosed in the present description, can be used as molecular markers in the method of crossing using a marker (MAB). The DNA molecules of the present invention can be used in methods (such as AFLP markers, RFLP markers, RAPD markers, SNPs and SSRs) that identify genetically linked agronomically useful traits that are known in the art. A sign of herbicide resistance can be traced in the progeny from crossing with a corn plant according to the present invention (or its progeny and any other corn variety) using the MAB methods. DNA molecules are markers of such a trait, and MAB methods that are well known in the art can be used to trace the herbicide resistance trait (s) in maize plants, with at least one maize line of the present invention or its progeny is a parent or ancestor. The methods of the present invention can be used to identify any kind of corn in which this event takes place.

The methods of the present invention include a method for producing a herbicide resistant maize plant, said method comprising crossbreeding with a plant for use in the present invention. More specifically, these methods may include crossing two plants according to the present invention or one plant according to the present invention and any other plant. Preferred methods further include selecting offspring from said crosses as a result of analyzing said offspring with respect to an event detected according to the present invention. For example, the present invention may include tracking this event during cross-breeding cycles with plants having other desired characteristics, such as agronomic characteristics, such as those evaluated in the present description in various examples. Plants in which this event takes place and which have the required trait can be identified, identified, selected and promptly used, for example, in further rounds of crosses. This event / sign can also be combined throughout the crosses and traced according to the present invention with a sign (s) of resistance to insects and / or with additional signs of resistance to herbicides. One preferred variant of such traits of resistance to herbicides is a plant having this event in combination with a gene encoding resistance to dicamba herbicide.

In addition, AAD-1 alone or in combination with one or more additional features of HTC may be stacked with one or more additional features introduced (e.g., insect resistance, fungus resistance or stress resistance, etc.) or symptoms associated with the resulting product (for example, increased productivity, improved oil profile, improved fiber quality, etc.). Thus, the present invention can be used to obtain a complete agronomic complex of improved crop quality with the possibility of flexible and cost-effective control of any number of agricultural pests.

Methods for integrating a polynucleotide sequence into a specific chromosome region of a plant cell as a result of homologous recombination have been described in the art. For example, site-specific integration is described in US Patent Application Publication No. 2009/0111188 A1, which describes the use of recombinases or integrases to mediate the introduction of a donor polynucleotide sequence into a chromosome target. In addition, international patent application No. WO 2008/021207 describes zinc finger-mediated homologous recombination for integrating one or more donor polynucleotide sequences into specific positions of the genome. The use of recombinases, such as FLP / FRT, which are described in US Pat. No. 6,720,475, or CRE / LOX, which are described in US. Pat. No. 5,658,772, can be used to integrate the polynucleotide sequence into a particular region of the chromosome. Finally, the use of meganucleases for the targeted introduction of donor polynucleotides to a specific position on the chromosome is described by Puchta et al., PNAS USA 93 (1996) p. 5055-5060.

Other various methods of site-specific integration into plant cells are generally known and can be used (Kumar et al., Trands in Plant Sci. 6 (4) (2001) pp. 155-159). In addition, site-specific recombination systems that have been identified in several prokaryotic and lower eukaryotic organisms can be used in plants. Examples of such systems include, but are not limited to: the R / RS recombinase system from the plasmid pSR1 of the yeast Zygosaccharomyces rouxii (Araki et al. (1985) J. Mol. Biol. 182: 191-203) and the Gin / gix system of the phage Mu (Maeser and Kahlmann (1991 ) Mol. Gen. Genet. 230: 170-176).

In some embodiments of the present invention, it may be desirable to integrate or stack new transgene (s) in the vicinity of an existing transgenic event. A transgenic event can be considered, the preferred genomic locus of which was selected based on unique characteristics such as a single insertion site, normal Mendelian cleavage and stable expression, and an excellent combination of efficacy, including herbicide resistance and agronomic efficacy in many locations with different environmental conditions. Newly integrated transgenes should preserve the transgene expression characteristics of existing transformants. In addition, analyzes can be developed to identify and confirm a new integration event, since genomic flanking sequences and the position in the chromosome of a new integration event have already been identified. Finally, integrating a new transgene into a specific position on the chromosome that is linked to an existing transgene can simplify the introgression of transgenes into another genetic background as a result of sexual outcrossing using conventional cross-breeding methods.

In some embodiments, it may be desirable to excise polynucleotide sequences from a transgenic insert. For example, transgene excision as described in provisional application for the grant of US patent No. 61/297628, which describes the use of nuclei with zinc fingers to remove a polynucleotide sequence consisting of an expression cassette from an integrated chromosome as a result of a transgenic insertion event. The polynucleotide sequence that is deleted may be a selectable marker. After excision and deletion of the polynucleotide sequence, the modified transgenic event can be re-targeted using the insertion of the polynucleotide sequence. Cutting out the polynucleotide sequence and subsequent re-targeted implementation of the modified transgenic event provides benefits such as reusing a selectable marker or overcoming unforeseen changes in the plant transcript resulting from the expression of specific genes.

In the present description, a specific region in chromosome 2 in the maize genome is disclosed which is very good for insertion of heterologous nucleic acids. Also disclosed is a molecular 5'-marker, molecular 3'-marker, 5'-flanking sequence and 3'-flanking sequence, applicable to identify the position of the target region in chromosome 2. Thus, the present invention relates to methods of introducing heterologous nucleic acids of interest acids in or near such a predetermined target region. The present invention also encompasses the use of a corn seed and / or corn plant containing any heterologous nucleotide sequence inserted in or near the claimed target region. One embodiment of such targeted integration is to cut and / or replace with another insert instead of the pat expression cassette, exemplified herein. In this regard, according to the present invention can be used, for example and without limitation, targeted homologous recombination.

As used herein, “stacking” of genes, events, or traits means combining the desired traits in a single transgenic line. Plant breeders stack the transgenic traits by interbreeding between the parents, each of which has the desired trait, and then identifies the offspring that possesses both of the required traits. Another way of gene stacking is the transfer during the transformation of two or more genes into the nucleus of a plant cell at the same time. Another way of stacking genes is to re-transform the transgenic plant into another gene of interest. For example, gene stacking can be used to combine two or more different traits, including, for example, two or more different insect traits, insect resistance trait (s) and disease resistance trait (s), two or more herbicide resistance traits and / or insect resistance sign (s) and herbicide resistance sign (s). The use of a selectable marker in addition to the gene of interest can also be considered gene stacking.

“Homologous recombination” refers to the interaction between any pair of nucleotide sequences having corresponding regions containing a similar nucleotide sequence by which two nucleotide sequences can interact (recombine) to form a new recombinant DNA sequence. Each of the sites with a similar nucleotide sequence is referred to herein as a “homology sequence." In general, the frequency of homologous recombination increases with increasing homology sequence length. Thus, although homologous recombination can occur between two nucleotide sequences whose length is shorter than the length of an identical sequence, the frequency of recombination (or efficiency) decreases as the divergence between the two sequences increases. Recombination can be carried out using one homology sequence in the donor molecule and the target molecule, while creating a recombination product resulting from a “single crossing-over”. Alternatively, two homology sequences can be placed in each of the nucleotide sequences: the donor sequence and the target sequence. Recombination between two homology sequences in a donor with two homology sequences in a target generates a recombination product resulting from a double crossing over. If the homology sequences in the donor molecule flank the sequence that needs to be processed (for example, the sequence of interest), then recombination due to double crossing over with the target molecule will result in a recombination product in which the sequence of interest replaces the DNA sequence that was originally located between homology sequences in the target molecule. The exchange of DNA sequences between a target and a donor as a result of a recombination event due to double crossing over is called "sequence replacement."

The AAD-1 enzyme under consideration provides the possibility of transgenic expression leading to resistance to combinations of herbicides that would provide the ability to control almost all broad-leaved and cereal weeds. AAD-1 can serve as an excellent feature of a herbicide-resistant crop (HTC), for example, for stacking with other HTC traits (e.g. glyphosate resistance, glufosinate resistance, imidazolinone resistance, bromoxynil resistance, etc.) and signs of insect resistance (Cry1F, Cry1Ab, Cry 34/45, etc.). In addition, AAD-1 can serve as a selectable marker to facilitate the selection of primary plant transformants genetically engineered with a second gene or group of genes.

The HTC features of the present invention can be used in new combinations with other HTC features (including but not limited to glyphosate resistance). Such combinations of traits provide new ways of controlling weed (and the like) species due to newly acquired resistance or hereditary resistance to herbicides (e.g. glyphosate). Thus, in addition to the features of HTC, new methods of weed control using herbicides to which resistance has been obtained in transgenic cultivated plants due to this enzyme are included in the scope of the invention.

In addition, glyphosate-resistant crops grown around the world are widespread. Repeated alternation with other glyphosate-resistant crops, control of glyphosate-resistant self-sowing plants may be difficult in the case of alternating crops. Thus, the use of transgenic traits according to the present invention, individually stacked or transformed into cultivated plants, provides a means of controlling other self-seeding HTC cultures.

A preferred plant or seed for use in the present invention contains in its genome the inserted sequences that are described in the present description, together with at least 20-500 or more consecutive flanking nucleotides on both sides of the insert that are identified in the present description. Unless otherwise indicated, reference to flanking sequences refers to sequences determined in relation to the sequence of SEQ ID NO: 29 (see table above). In turn, the sequence of SEQ ID NO: 29 includes heterologous DNA inserted into the original transformant, and illustrative flanking genomic sequences immediately adjacent to the inserted DNA. It can be expected that all or part of such flanking sequences can be passed on to offspring that receive the inserted DNA, including the transgene of interest, as a result of sexually crossing the same parental line in which such an event occurred.

The present invention relates to the use of tissue cultures from regenerated plant cells according to the present invention. The invention also relates to the use of a plant regenerated from such a tissue culture, especially when said plant is capable of expressing all the morphological and physiological properties of the variety used as an example. Preferred plants for use in the present invention may possess all the physiological and morphological properties of a plant grown from deposited seed. The present invention further relates to the use of offspring of such a seed and to a seed having qualitative properties of interest.

Manipulation (such as mutation, additional transfection and further crossbreeding) with plants or seeds or parts thereof can lead to the creation of so-called “essentially derived” varieties. The International Union for the Protection of New Varieties of Plants (UPOV) has provided the following guidance on determining whether a variety is essentially derived from a protected variety:

[A] A variety shall be deemed to be essentially derived from another variety (“the original variety”) if:

(i) it is predominantly produced from the original variety or from a variety that itself is mainly produced from the original variety, while maintaining the manifestation of the main characteristics resulting from the implementation of the genotype or combination of genotypes of the original variety;

(ii) it is clearly different from the original variety; and

(iii) with the exception of differences that are due to origin, it corresponds to the original variety in the degree of manifestation of the main characteristics resulting from the implementation of the genotype or combination of genotypes of the original variety.

UPOV, Sixth Meeting with International Organizations, Geneva, Oct. 30, 1992; document prepared by the Bureau of the Union.

As used herein, “line” means a group of plants in which there is little or no genetic variation among individuals with respect to at least one trait. Some lines can be created as a result of self-pollination in several generations and selection or as a result of vegetative propagation of the original parent using tissue or cell culture techniques.

As used in the present description, the terms “cultivar” and “cultivar” are synonyms and refer to the line used for industrial production.

"Stability" or "stable" in relation to this component means that the component is maintained from generation to generation and, preferably, for at least three generations at essentially the same level, for example, preferably ± 15%, more preferably ± 10%, most preferably ± 5%. Stability can be affected by temperature, location, stress, and sowing time. A comparison of subsequent generations in the field should show that the component is present in a similar way.

“Commercial value” is defined as the presence of good plant power and high fertility, so that the crop can be used by farmers in production using conventional agricultural equipment, and oil containing the described components can be extracted from the seed using conventional crushing and extraction equipment. To be commercially viable, yields measured by seed weight, oil content, and total oil per acre (0.4 ha) should be within 15% of the average yield of an otherwise comparable commercial variety corn without improved valuable properties grown in the same region.

“Agronomically elite” means that the line has the required agronomic characteristics, such as yield, maturity, disease resistance and the like, in addition to insect resistance due to the event (s) according to the invention. Agronomic characteristics, taken individually or in any combination, which are indicated in the examples below, in the plant in which the event of the present invention occurred, are included in the scope of the present invention. Any or all of the agronomic characteristics and measured parameters can be used to identify such plants, either as a point or at either end or both ends of the range of characteristics used to identify such plants.

As will be appreciated by one of ordinary skill in the art in light of the present description, preferred detection kit variants, for example, may contain probes and / or primers directed to “connection sequences” and / or containing “connection sequences” or “transition sequences” (where flanking the genomic sequence of maize converges with the insertion sequence). For example, a kit contains polynucleotide probes, primers and / or amplicons designed to identify one or both sequences of a compound (where the insert converges with the flanking sequence), which are shown in Table 1. One general idea is to have one primer that hybridizes with a flanking region, and one primer that hybridizes with the insert. Each of these primers often has a length of at least ~ 15 residues. With this arrangement, primers can be used to form / amplify a detectable amplicon, which indicates the presence of an event according to the present invention. Such primers can be used to form an amplicon that encompasses (and includes) the sequence of the compound as described above.

The primer (s) that "sits" (s) on the flanking sequence is designed so that it does not hybridize within more than about 200 bases or outside the junction. Thus, typical flanking primers can be designed containing at least 15 residues of any yarn within 200 bases in the flanking sequences from the start of the insertion. That is, primers containing a sequence of an appropriate size within residues ~ 1674-1873 and / or ~ 6690-6890 of SEQ ID NO: 29 are included in the scope of the present invention. Similarly, primers for insertion can be designed to any location on the insert, and for such designing primers can be used without exception, for example, residues ~ 1874-2074 and ~ 6489-6689.

One skilled in the art will also understand that primers and probes for hybridization can be designed in the range of standard hybridization conditions and / or PCR with a portion of the sequence SEQ ID NO: 29 (or complement) and its complements, while the primer or probe is not absolutely complementary to an exemplary sequence. That is, some degree of erroneous mating may be acceptable. In the case of a primer of, for example, approximately 20 nucleotides in length, usually about one or two nucleotides do not have to bind to the opposite strand if the mismatched base is internal or is located at the end of the primer that is opposite to the amplicon. The following are various suitable hybridization conditions. Synthetic nucleotide analogs, such as inosine, can also be used as probes. You can also use probes based on peptide nucleic acids (PNA), as well as DNA and RNA probes. It is important that such probes and primers are diagnostic (capable of uniquely identifying and distinguishing) with respect to the presence of an event according to the present invention.

It should be noted that errors may occur in PCR amplification, which can lead, for example, to small sequencing errors. That is, unless otherwise indicated, the sequences listed in the present description were determined by creating long amplicons with the genomic DNA of the corn and then cloning and sequencing the amplicons. It is not unusual to find small differences and minor mismatches in the sequences formed and defined in this way, given the many rounds of amplification that are necessary to create an amplicon sufficient for sequencing from genomic DNA. The person skilled in the art should understand and bear in mind that any adjustments necessary due to common sequencing errors or mismatches of these types are included in the scope of the present invention.

It should also be noted that it is not unusual to delete some genomic sequences, for example, when a sequence is inserted in the course of a specific event. Thus, some differences may also appear between the flanking sequences under consideration and the genomic sequences listed, for example, in GENBANK. Some of these differences are discussed below in the Examples section. Accordingly, probes and primers can be adjusted.

Thus, the use of a plant containing a polynucleotide that has a certain degree of identity with the flanking and / or insert sequences of the invention is within the scope of the present invention. The sequence identity of the present invention may mean that the polynucleotide sequence has at least 65% sequence identity, preferably at least 70% sequence identity, more preferably at least 75% sequence identity, more preferably at least 80% identity, and more preferably at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with sequence given in ETS example or described in this publication. Hybridization and hybridization conditions, which are proposed in the present invention, can also be used to determine such plants and polynucleotide sequences according to the present invention. The sequence of flanking sequences plus insertion sequences can be confirmed with reference to the deposited seed.

The components of each “insert” are shown in figures 1 and 2 and are discussed in more detail below in the examples. Polynucleotide DNA sequences of such components or fragments thereof can be used as DNA primers or probes in the methods of the present invention.

In some embodiments, compositions and methods are provided for detecting the presence of a transgene / genomic insertion region in plants and seeds and the like of a corn plant. DNA sequences are provided that comprise the transgene / genomic insertion sequence of the present invention (between residues 1873-1874 and 6689-6690 of SEQ ID NO: 29), fragments thereof and complements of exemplary sequences and any fragments thereof. The sequence of connection of the insertion region encompasses the connection between heterologous DNA integrated into the genome and DNA from maize cells flanking the insertion site. Such sequences may be diagnostic for a given event.

Based on such insertion sequences and boundary sequences, event-specific primers can be created. PCR analysis showed that the maize lines of the present invention can be identified in different maize genotypes in the analysis of PCR amplicons generated using such event-specific primer sets. These and other related methods can be used to uniquely identify such lines of corn. Thus, PCR amplicons obtained using such primer pairs are unique and can be used to identify such maize lines.

In some embodiments, sequences that contain a continuous fragment of a new transgene / genomic insertion region are an aspect of the present invention. The scope of the invention includes DNA sequences that contain polynucleotides of sufficient transgene insertion sequence length and polynucleotides of sufficient length of the genomic sequence of maize from one or more of the three above corn plants and / or sequences that are applicable as primer sequences to obtain the product in the form of an amplicon, which is diagnostic for one or more of these corn plants.

Related variants relate to DNA sequences that contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more consecutive nucleotides of the transgenic part of the DNA sequence described herein (such as SEQ ID NO: 29 and its portions), or its complement and flanking corn DNA sequence of similar length from such sequences or their complements. Such sequences are useful as DNA primers in DNA amplification methods. Amplicons obtained using such primers are diagnostic for any events occurring in the corn plants described herein. Thus, the invention also relates to amplicons obtained using such DNA primers and homologous primers.

The present invention may also relate to methods for detecting the presence of DNA in a sample that corresponds to an event in a corn plant as described herein. Such methods may include: (a) contacting a sample containing DNA with a set of primers that, when used in an amplification reaction of nucleic acid with DNA from at least one of these corn events, produces an amplicon that is diagnostic for the specified event (s) ; (b) carrying out a nucleic acid amplification reaction, thereby producing an amplicon; and (c) detection of amplicon.

Additional detection methods may include a method for detecting the presence of DNA in a sample corresponding to at least one of these events, said method comprising: (a) contacting the sample containing DNA with a probe that hybridizes under stringent hybridization conditions with at least DNA at least one of the indicated events in corn and which does not hybridize under stringent conditions of hybridization with the DNA of the control corn plant (DNA that is not related to the event of interest); (b) placing the sample and probe under stringent hybridization conditions; and (c) detecting probe hybridization with DNA.

Some options include methods for producing a corn plant in which the aad-1 event of the present invention takes place, said method comprising the steps of: (a) sexually crossing the first parent line of the corn (containing the expression cassette of the present invention, which confers the indicated resistance to herbicides to plants of the indicated line) and the second parent line of corn (in which there is no such sign of resistance to herbicides) with the receipt of many descendant plants; and (b) selection of progeny plants using molecular markers. Such methods may optionally include an additional step of crossbreeding the progeny plants with the second parent line of the corn to produce a high-grade corn plant that has the indicated insect resistance trait.

According to another aspect of the invention, methods are provided for determining the zygosity of offspring from interbreeding with any one (or more) of these three events. These methods may include contacting a sample containing maize DNA with the primer kit of the present invention. These primers when used in the amplification reaction of nucleic acids with genomic DNA from at least one of the indicated events of the corn give the first amplicon, which is diagnostic for at least one of these events of the corn. Such methods further include carrying out a nucleic acid amplification reaction to produce a first amplicon; detection of the first amplicon; and making contact of the sample containing corn DNA with the specified set of primers (the specified set of primers when used in the amplification reaction of nucleic acid with genomic DNA from maize plants gives a second amplicon containing the native genomic DNA of the corn homologous to the region of the maize genome); and carrying out a nucleic acid amplification reaction to obtain a second amplicon. The methods further include detecting a second amplicon and comparing the first and second amplicons in the sample, wherein the presence of both amplicons indicates that the sample is heterozygous for insertion of the transgene.

DNA detection kits can be developed using the compositions disclosed herein and methods well known in the field of DNA detection. The kits are useful for identifying in a sample DNA associated with an event in maize according to the invention, and may be useful in methods for breeding maize plants containing such DNA. The kits contain DNA sequences homologous or complementary to amplicons, for example, disclosed in the present description, or DNA sequences homologous or complementary to the DNA located in the genetic elements of the transgene in the event of an event according to the invention. Such DNA sequences can be used in DNA amplification reactions or as probes in a DNA hybridization method. Kits may also contain reagents and materials necessary for the implementation of the detection method.

“Probe” means an isolated nucleic acid molecule to which a common detectable label or reporter molecule is associated (such as a radioactive isotope, ligand, chemiluminescent agent or enzyme). Such a probe is complementary to the target nucleic acid strand, in the case of the present invention, the genomic DNA strands of one of these events in corn, either from a corn plant or from a sample that contains DNA resulting from such an event. Probes according to the present invention include not only deoxyribonucleic or ribonucleic acids, but also polyamides and other substances for probes that specifically bind to the target DNA sequence and can be used to detect the presence of such a target DNA sequence.

"Primers" are isolated / synthesized nucleic acids that are annealed with a complementary strand of a target DNA during hybridization of the nucleic acids to form a hybrid between the primer and the strand of the target DNA, and then extended along the strand of the target polymerase, for example, DNA polymerase. The primer pairs of the present invention are useful for amplification of a target nucleic acid sequence, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

Probes and primers typically have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 , 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102 , 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 , 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152 , 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 , 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202 , 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413,414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 nucleotides or more. Such probes and primers specifically hybridize to the target sequence under high stringency conditions. Preferably, the probes and primers according to the present invention have complete sequence similarity with the target sequence, although probes can be constructed using conventional methods that are different from the target sequence and retain the ability to hybridize with the target sequences.

Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2 ^nd ed., Vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Pairs of PCR primers can be obtained from a known sequence, for example, using computer programs designed for this purpose.

Primers and probes based on the flanking DNA sequences and inserts disclosed herein can be used to confirm (and, if necessary, adjust) the described sequences by conventional means by re-cloning and sequencing such sequences.

The nucleic acid probes and primers of the present invention hybridize under stringent conditions to the target DNA sequence. You can use any conventional method of hybridization or amplification of nucleic acids to identify the presence in the sample of DNA inserted as a result of a transgenic event. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain conditions. In the sense used in the present description, it is said that two nucleic acid molecules are capable of specifically hybridizing with each other if the two molecules are capable of forming a nucleic acid structure of two antiparallel strands. It is said that a nucleic acid molecule is a “complement” of another nucleic acid molecule if their full complementarity is shown. In the sense used in the present description, it is said that the molecules have “full complementarity” when each nucleotide of one of the molecules is complementary to the nucleotide of the other. Molecules are said to be “minimally complementary” if they can hybridize with each other with stability sufficient to ensure that they remain annealed with each other at least under normal “low stringency” conditions. Similarly, it is said that molecules are “complementary” if they can hybridize with each other with stability sufficient to ensure that they remain annealed to each other under normal “high stringency” conditions. The usual stringency conditions are described by Sambrook et al., 1989. Deviations from full complementarity are acceptable, provided that such deviations do not completely exclude the ability of the molecules to form a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe, it must only be sufficiently complementary in sequence so that it can form a stable double-stranded structure in the particular solvent used and at the salt concentrations used.

As used herein, a substantially homologous sequence is a nucleic acid sequence that will specifically hybridize to a complement of the nucleic acid sequence with which it is compared under high stringency conditions. The term "stringent conditions" is functionally defined in relation to the hybridization of a probe nucleic acid with a target nucleic acid (i.e., with a particular nucleic acid sequence of interest) using the specific hybridization method discussed in Sambrook et al., 1989, sections 9.52-9.55. See also Sambrook et al., 1989, sections 9.47–9.52 and 9.56–9.58. Accordingly, the nucleotide sequences of the invention can be used in connection with their ability to selectively form duplex molecules with complementary regions of DNA fragments.

Depending on the intended application, different hybridization conditions can be used to achieve different degrees of selectivity of the probe with respect to the target sequence. For applications requiring high selectivity, relatively stringent conditions for the formation of hybrids can usually be used, for example, conditions of relatively low salt concentration and / or high temperature, such as conditions created at a NaCl concentration of from about 0.02 M to about 0, can be selected. , 15 M at temperatures from about 50 ° C to about 70 ° C. Severe conditions, for example, may include washing the hybridization filter at least twice with high rigidity washing buffer (0.2X SSC, 0.1% SDS, 65 ° C). Suitable stringency conditions that stimulate DNA hybridization, e.g. 6.0X sodium chloride / sodium citrate (SSC) at about 45 ° C followed by a 2.0 × SSC wash at 50 ° C, are known to those skilled in the art, 6.3.1-6.3 .6. For example, the salt concentration in the washing step can be selected from conditions ranging from low hardness of about 2.0 × SSC at 50 ° C to high hardness of about 0.2 × SSC at 50 ° C. In addition, the temperature in the washing step can be increased from low hardness conditions at room temperature of about 22 ° C to high hardness conditions at about 65 ° C. Both temperature and salt can vary, either temperature or salt concentration can be kept constant, while another variable is changed. Such selective conditions to a small extent or generally do not allow inconsistencies between the probe and the matrix or the target thread. The identification of DNA sequences using hybridization is well known to specialists in this field, and the instructions given in US patent No. 4965188 and 5176995, are examples of methods of analysis of hybridization.

In some embodiments, the nucleic acid for use in the present invention will specifically hybridize to one or more primers (or amplicons or other sequences) provided as examples or provided herein, including their complement and fragments, under high stringency conditions. In one aspect, the nucleic acid marker molecule of the present invention has the nucleic acid sequence indicated in SEQ ID NO: 3-14, or its complements and / or fragments.

In another aspect, the nucleic acid marker molecule of the present invention has from 80% to 100%, or from 90% to 100% sequence identity with such nucleic acid sequences. In a further aspect, the nucleic acid marker molecule for use in the present invention has from 95% to 100% sequence identity with that sequence. Such sequences can be used as markers in plant breeding methods to identify offspring from genetic crosses. Hybridization of the probe with the target DNA molecule can be detected by any of a number of methods known to those skilled in the art, and such methods include, but are not limited to, fluorescent labels, radioactive labels, antibody-based labels, and chemiluminescent labels.

Regarding amplification of a target nucleic acid sequence (for example, in PCR) using a specific pair of amplification primers, “stringent conditions” are conditions that allow hybridization of a pair of primers only to the target nucleic acid sequence with which the primer having the corresponding sequence wild-type (or its complement), can bind and preferably give a unique amplicon amplification product.

The term “specific for (target sequence)” indicates that the probe or primer hybridizes under stringent hybridization conditions only to the target sequence in the sample containing the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product of amplification of a nucleic acid of a target nucleic acid sequence that is part of a nucleic acid matrix. For example, in order to determine if a corn plant obtained by sexual intercourse contains the genomic DNA of a transgenic event from a corn plant according to the present invention, DNA from a tissue sample of a corn plant can be subjected to nucleic acid amplification by a method using a pair of primers that includes a primer obtained from a flanking sequence located in the plant’s genome near the insertion site of the inserted heterologous DNA, and a second primer obtained from the inserted hetero biological DNA to produce an amplicon, which is diagnostic for the presence of DNA inserted as a result of that event. Amplicon has a specific length and sequence, which are also diagnostic for this event. The amplicon may have a length ranging from the total length of the primer pair plus one base pair of nucleotides and / or the total length of the primer pair plus approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 , 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 , 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 , 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 , 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 , 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188 , 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 , 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238 , 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263 , 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288 , 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313 , 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338 , 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363 , 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388 , 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413 , 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438 , 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463 , 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488 , 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500, 750, 1000, 1250, 1500, 1750, 2000 or more base pairs (plus or minus any number of the above ) Alternatively, a pair of primers can be obtained from a flanking sequence located on both sides of the inserted DNA, so as to obtain an amplicon that contains the entire nucleotide sequence of the insert. A representative pair of primers obtained from the genomic sequence of a plant can be located at some distance from the sequence of the inserted DNA. Such a distance can range from one base pair of nucleotides up to about twenty thousand base pairs of nucleotides. The use of the term "amplicon", in particular, excludes dimers of primers that can be formed in the thermal reaction of DNA amplification.

Amplification of nucleic acids can be carried out by any of various nucleic acid amplification methods known in the art, including polymerase chain reaction (PCR). Numerous amplification methods are known in the art, and they are described along with other publications in US Pat. No. 4,683,195 and US Pat. No. 4,683,202. PCR amplification methods have been developed for amplification of up to 22 kb. genomic DNA. Such methods, as well as other methods known in the field of DNA amplification, can be used in the practical implementation of the present invention. The heterologous transgenic DNA insertion sequence or flanking genomic sequence for a given corn event can be confirmed (and adjusted if necessary) by amplification of such sequences associated with this event using primers derived from the sequences of the present invention followed by standard sequencing DNA of a PCR amplicon or cloned DNA.

The amplicon obtained by these methods can be detected using many methods. Agarose gel electrophoresis and ethidium bromide staining is a well-known method for detecting amplicon DNA. Another such method is the analysis of units of genetic information (Genetic Bit Analysis), in which case DNA oligonucleotides are constructed that overlap both the nearby flanking genomic DNA sequence and the integrated DNA sequence. Oligonucleotides are immobilized in the wells of a microtiter plate. After the PCR of the region of interest (using one primer for the inserted sequence and one for the adjacent flanking genomic sequence), the single-stranded PCR product can be hybridized with an immobilized oligonucleotide and can serve as a template for a one-base extension reaction using DNA polymerase and labeled ddNTPs specific for the expected next base. Data recording can be based on fluorescence or based on ELISA. The signal indicates the presence of an insert / flanking sequence, taking into account successful amplification, hybridization, and extension to one base.

Another method is a pyrosequencing method as described by Winge (Innov. Pharma. Tech. 00: 18-24, 2000). In the case of this method, an oligonucleotide is constructed that overlaps the connection of the adjacent genomic DNA and the insert DNA. The oligonucleotide is hybridized with a single-stranded PCR product from the region of interest (one primer for the inserted sequence and one for the flanking genomic sequence) and incubated in the presence of DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine-5’-phosphosulfate and luciferin. dNTP is added separately, and inclusion leads to the appearance of a light signal, which is measured. A light signal indicates the presence of a transgenic insert / flanking sequence, taking into account successful amplification, hybridization, and extension to one or many bases.

Fluorescence polarization is another method that can be used to detect the amplicon of the present invention. Following this method, an oligonucleotide is constructed that encompasses a compound of genomic flanking DNA and integrated DNA. The oligonucleotide is hybridized with a single-stranded PCR product from the region of interest (one primer for the integrated DNA and one for the flanking sequence of the genomic DNA) and incubated in the presence of DNA polymerase and fluorescently-labeled ddNTP. Elongation by one base leads to the inclusion of ddNTP. Inclusion can be measured as a change in polarization using a fluorimeter. A change in polarization indicates the presence of a transgenic insert / flanking sequence, taking into account successful amplification, hybridization, and elongation on one base.

TAQMAN (PE Applied Biosystems, Foster City, Calif.) Is a method for detecting and quantifying the presence of a DNA sequence. Briefly, an FRET oligonucleotide probe is constructed that overlaps the connection of the genomic flanking DNA and the insert DNA. The FRET probe and PCR primers (one primer for the insert DNA sequence and one for the flanking genomic sequence) undergo a cyclic reaction in the presence of thermostable polymerase and dNTP. During specific amplification, Taq DNA polymerase removes and releases the fluorescent residue from the bond with the quenching residue on the FRET probe. A fluorescence signal indicates the presence of a flanking sequence / transgenic insert sequence, taking into account successful amplification and hybridization.

The molecular beacon method is described for use in sequence detection. Briefly, an FRET oligonucleotide probe is constructed that overlaps the connection of flanking genomic DNA and insert DNA. The result of the unique structure of the FRET probe is that it has a secondary structure that keeps the fluorescent residue and quenching residue in close proximity. The FRET probe and PCR primers (one primer for the insert DNA sequence and one for the flanking genomic sequence) undergo a cyclic reaction in the presence of thermostable polymerase and dNTP. After successful PCR amplification, hybridization of the FRET probe with the target sequence leads to the elimination of the secondary structure of the probe and the spatial separation of the fluorescent and quenching residues. The fluorescence signal indicates the presence of a flanking genomic / transgenic insert sequence, taking into account successful amplification and hybridization.

In addition to revealing a position in the maize genome that is excellent for insertion, the present invention also relates to the use of a maize seed and / or maize plant containing at least one insert unrelated to aad-1 near such a position in the genome. One option is to replace another insert instead of the aad-1 insert, which is given as an example in the present description. In general, according to the present invention, for example, targeted homologous recombination can be used. A technique of this type is, for example, the subject of application WO 03/080809 A2 and the corresponding published application for the grant of a US patent (US20030232410). Thus, the present invention relates to the use of plants and plant cells containing a heterologous insert (instead of or simultaneously with multiple copies of aad-1) flanked by all or a known part of the flanking sequences described herein (e.g., residues 1-1873 and 6690- 8557 of SEQ ID NO: 29).

The following are some points / variants of plants, seeds, polynucleotides and detection methods that can be used according to some variants of implementation of the present invention:

1. A transgenic corn plant containing a genome, wherein said genome contains the sequence of SEQ ID NO: 29.

2. A corn seed containing the genome in which the AAD-1 event DAS-40278-9 occurs, which is present in a seed deposited in the American Type Culture Collection (ATCC) with accession number PTA-10244.

3. The corn seed according to claim 2, in which the specified seed contains the genome, and the specified genome contains the sequence of SEQ ID NO: 29.

4. A corn plant obtained by growing from a seed according to claim 2, wherein said plant contains the sequence of SEQ ID NO: 29.

5. A descendant plant obtained from a corn plant according to claim 4, wherein in said descendant plant there is an AAD-1 event DAS-40278-9.

6. Herbicide-resistant offspring obtained from a corn plant according to claim 1, wherein said offspring contains the sequence of SEQ ID NO: 29.

7. A transgenic maize plant containing a transgenic insert in a portion of the maize chromosome that is a target located on chromosome 2 at a position of about 20 cM between the SSR markers of UMC1265, partially amplified using the sequences SEQ ID NO: 30 and SEQ ID NO: 31, and MC0111, partially amplified using the sequences of SEQ ID NO: 32 and SEQ ID NO: 33, wherein the target region contains a heterologous nucleic acid.

8. The method for producing a transgenic corn plant according to claim 7, wherein said method comprises inserting a heterologous nucleic acid into a position on chromosome 2 of about 20 cM between the SSR markers of UMC1265, partially amplified using the sequences SEQ ID NO: 30 and SEQ ID NO: 31, and MMC0111, partially amplified using the sequences of SEQ ID NO: 32 and SEQ ID NO: 33.

9. The plant part according to claim 4, wherein said part is selected from the group consisting of pollen, ovule, flowers, seed boxes, fibers, shoots, roots and leaves, while this part contains the sequence of SEQ ID NO: 29.

10. The transgenic corn plant according to claim 7, wherein said plant contains a transgene insert in the genomic sequence or flanked by a genomic sequence selected from the group consisting of residues 1-1873 of the sequence SEQ ID NO: 29 and residues 6690-8557 of the sequence SEQ ID NO : 29.

11. An isolated polynucleotide molecule, wherein said molecule:

contains at least 15 nucleotides and supports hybridization under stringent washing conditions with a nucleic acid sequence selected from the group consisting of residues 1-1873 of the sequence SEQ ID NO: 29, residues 6690-8557 of the sequence SEQ ID NO: 29 and their complements;

contains a nucleotide sequence selected from the group consisting of sequences of SEQ ID NO: 1-33; and / or

hybridizes under stringent washing conditions with a nucleotide sequence selected from the group consisting of residues 1863-1875 of the sequence SEQ ID NO: 29, residues 6679-6700 of the sequence SEQ ID NO: 29 and their complements.

12. The isolated polynucleotide of claim 11, wherein said polynucleotide contains a nucleotide sequence selected from the group consisting of the sequences SEQ ID NO: 1-33.

13. The isolated polynucleotide of claim 11, wherein said polynucleotide hybridizes under stringent washing conditions with a nucleotide sequence selected from the group consisting of residues 1863-1875 of SEQ ID NO: 29, residues 6679-6700 of SEQ ID NO: 29, and their complements.

14. The polynucleotide of claim 13, wherein said polynucleotide is an amplicon formed in a polymerase chain reaction.

15. A method for detecting an event in a maize sample containing maize DNA, said method comprising contacting said sample with at least one polynucleotide that is diagnostic for the AAD-1 maize DAS-40278-9 event that occurs in a seed, deposited at the American Type Culture Collection (ATCC) with accession number PTA-10244.

16. The method of claim 15, wherein said method comprises contacting said sample with

a) a first primer that binds to a flanking sequence selected from the group consisting of residues 1-1873 of the sequence SEQ ID NO: 29, residues 6690-8557 of the sequence SEQ ID NO: 29 and their complements; and

b) a second primer that binds to the insert sequence containing residues 1874-6689 of the sequence SEQ ID NO: 29 or its complement;

subjecting said polymerase chain reaction sample; and analysis for an amplicon formed between said primers.

17. The method according to clause 16, in which these primers are selected from the group consisting of sequences of SEQ ID NO: 1-28.

18. The method according to clause 15, wherein said polynucleotide contains at least 30 nucleotides and hybridizes under stringent conditions with a sequence selected from the group consisting of residues 1863-1875 of the sequence SEQ ID NO: 29, residues 6679-6700 of the sequence SEQ ID NO: 29 and their complements; wherein said method further comprises subjecting said sample and said hybridization polynucleotide to stringent conditions; and analyzing said sample with respect to hybridization of said polynucleotide with said DNA.

19. A kit for detecting DNA containing the first primer and the second primer according to claim 17.

20. A kit for detecting DNA, designed to implement the method according to p.

21. A kit for detecting DNA containing the polynucleotide according to item 13.

22. The polynucleotide of claim 12, wherein said polynucleotide comprises the sequence of SEQ ID NO: 29.

23. The method of producing polynucleotide according to item 22.

24. The method for producing a transgenic plant of claim 10, wherein said method comprises embedding a transgene in a DNA portion of a maize genome, said DNA portion having a 5 ′ end containing nucleotide residues 1-1873 of SEQ ID NO: 29, and 3 '' end containing nucleotide residues 6690-8557 of the sequence SEQ ID NO: 29.

25. A method comprising crossing a first corn plant containing the sequence of SEQ ID NO: 29 with a second corn plant to obtain a third corn plant containing the genome and analyzing said third corn plant with respect to the presence of the sequence SEQ ID NO: 29 in the specified genome.

26. The method according A.25, in which the method is used for breeding a corn plant and / or for introgression of the sign of resistance to herbicides in the corn plant.

All patents, patent applications, provisional applications, and publications referenced or cited in the present description are incorporated by reference to the extent that they do not contradict the detailed guidance given in the present description.

The following examples are included to illustrate the methods used in the practice of the invention and to demonstrate some preferred embodiments of the invention. Such examples should not be construed as limiting. Those skilled in the art will understand that the instructions given in the following examples represent specific approaches used to illustrate preferred methods for practicing the invention. However, in the light of the present description, it will be understood by those skilled in the art that numerous changes can be made to these specific options and still obtain similar or similar results without departing from the gist and without departing from the scope of the invention. Unless otherwise indicated, all percentages are by weight and all ratios in the solvent mixture are by volume unless otherwise noted.

The following examples include presowing and / or pre-emergence applications. Such applications are not limited to event "278". In addition, the applicability of the resistance provided by these AAD-1 genes can be explained taking into account the shortened period of time from application to new seeding. This provides plant growers with much greater flexibility in scheduling their crops relative to the moment of complete destruction of weeds (“burning”). Without the application of the present invention, waiting for 7-30 days or so after the destruction of the weeds before sowing can cause significant yield loss. Thus, the present invention provides advantages in this regard. See, for example, Example 13. Any intervals between sowing / applying herbicides and any concentration ranges / application rates of the herbicide (s) given as examples or proposed in the present description can be used according to the present invention.

Unless otherwise indicated, the following abbreviations are used.

Aad-1 aryloxyalkanoate dioxygenase-1 bp nucleotide pairs ° C degrees centigrade DNA Deoxyribonucleic acid Dig digoxygenin EDTA ethylenediaminetetraacetic acid tbp thousand pairs of nucleotides mcg micrograms μl microliter ml milliliter M molar mass OLP overlapping probe PCR polymerase chain reaction PTU plant transcriptional unit Sds sodium dodecyl sulfate Sop standard manufacturing operations SSC buffer solution containing a mixture of sodium chloride and sodium citrate, pH 7.0 TBE buffer solution containing a mixture of Tris base, boric acid and EDTA, pH 8.3 AT volts

EXAMPLES

Example 1. Transformation and selection of the event AAD1 pDAS 1740-278

Event AAD1, pDAS 1740-278, was obtained by the WHISKER-mediated transformation of the Hi-II maize line. The transformation method used is described in application for the grant of US patent No. 20090093366. The FspI fragment of plasmid pDAS1740, also called pDAB3812 (Figure 1), transformed the line of corn. The specified plasmid construct contains a plant expression cassette containing RB7 MARv3 :: promoter v2 ubiquitin 1 Zea mays // AAD1 v3 // 3’-UTR PER5 Zea mays :: transcriptional unit of the plant (PTU) RB 7 MARv4.

Received numerous events. Those events that were viable and resulted in a healthy haloxifope-resistant callus tissue were assigned unique identification codes representing the alleged transformation events and immediately transferred to fresh selection medium. Plants were regenerated from the tissue obtained as a result of each unique event and transferred to the greenhouse.

Leaf samples were taken for molecular analysis to confirm the presence of the AAD-1 transgene using a Southern blot, confirmation of DNA boundaries, and confirmation using genomic markers. Positive T0 plants were pollinated with inbred lines to obtain a T1 seed. T1 plants with event pDAS 1470-278-9 (DAS-40278-9) were selected, self-pollinated, and characterized for five generations. At the same time, T1 plants were backcrossed and introgressed into elite germplasm (XHH13) by selection using markers over several generations. Such an event was created on the basis of an independent transformed isolate. The event was selected based on its unique characteristics, such as a single insertion site, normal Mendelian cleavage and stable expression, and an excellent combination of efficacy, including herbicide resistance and agronomic efficacy in a wide range of genotypic backgrounds and in many locations with different environmental conditions. The following examples contain data that was used to characterize the event pDAS-1740-278-9.

Example 2. Characterization of the event pDAS 1740-278-9 using Southern blot

Southern blot analysis was used to establish the integration pattern of the inserted DNA fragment and determine the number of inserts / copies of the aad-1 gene in the event of pDAS-1740-278-9 (DAS-40278-9). Data was obtained to demonstrate the integration and integrity of the aad-1 transgene inserted into the maize genome.

Southern blot data indicate that insertion of the pDAS 1740 / Fsp I fragment in the event of DAS-40278-9 occurred in the simple integration of one intact copy of PTU aad-1 from plasmid pDAS1740. A detailed Southern blot analysis was performed using probes specific for the gene, promoter, terminator, and other regulatory elements located in the plasmid region and the restriction enzymes described that have cleavage sites located within the plasmid and produce hybridizing fragments within the plasmid or fragments that encompass the connection of the plasmid with the genomic DNA of maize (border fragments). The molecular masses shown on the basis of Southern hybridization data, in the case of a combination of this restriction enzyme and this probe, were unique for this event and made it possible to establish patterns for its identification. Such assays also showed that a plasmid fragment was inserted into the genomic DNA of maize without restructuring PTU aad-1. Identical hybridization fragments were observed in five different generations of corn in the case of the transgenic event DAS-40278-9, which indicates the stability of the inheritance of the PTU aad-1 insertion in generations. Hybridization using a mixture of three probes to the backbone located outside the Fsp I restriction site in pDAS1740 plasmid did not reveal any specific DNA / gene fragments, which indicates the absence of the ampicillin resistance gene and the absence of other regions of the vector backbone, directly near the restriction sites of the Fsp I plasmid pDAS1740 in the case of the transgenic event DAS-40278-9 of maize. An illustrative insert map in the case of aad-1-event DAS-40278-9 in corn is shown in figures 2, 3.

Example 2.1 Sample collection of maize leaves and isolation of genomic DNA (gDNA)

gDNA was obtained from the leaves of individual plants in which the corn aad-1 event DAS-40278-9 took place. The gDNA was extracted from leaf tissue collected from individual plants bearing the DAS-40278-9 maize aad-1 event. Used transgenic corn seeds from five different generations in the event of the event DAS-40278-9. Twenty individual corn plants, four plants per generation, were selected for event DAS-40278-9. In addition, gDNA was isolated from a conventional corn plant, XHH13, which contains a genetic background that is typical of the main line lacking the aad-1 gene.

Before isolation of gDNA from each plant, leaf-cut tissue samples were taken to test the expression of aad-1 protein using a set of test strips for rapid analysis (American Bionostica, Swedesboro, NJ) according to the method recommended by the manufacturer. Each sheet cut out by punching received a + or - score due to the presence or absence of aad-1, respectively. Only five-generation positive plants were subjected to further characterization in the event of the event DAS-40278-9.

Corn leaf samples were collected from individual plants in which the event DAS-40278-9, and the usual control XHH13 took place. Leaf samples were quickly frozen in liquid nitrogen and stored at about -80 ° C until use.

Individual genomic DNA was extracted from frozen maize leaf tissue following the standard CTAB method. If necessary, a certain amount of genomic DNA was further purified using Qiagen Genomic-Tip (Qiagen, Valencia, CA) in accordance with methods recommended by the manufacturer. After extraction, the amount of DNA was determined spectrofluorimetrically using a green Pico Green reagent (Invitrogen, Carlsbad, CA). Then the DNA was visualized on an agarose gel with confirmation of the values obtained in the analysis using Pico Green, and determining the quality of the DNA.

Example 2.2 DNA cleavage and separation

For molecular characterization of DNA, nine micrograms (9 μg) of genomic DNA from a DNA sample was split in the case of a corn DAS-40278-9 event and the usual control was added to each DNA sample from approximately five to eleven units of the selected restriction enzyme per microgram of DNA and the appropriate buffer for reactions. Each sample was incubated at approximately 37 ° C overnight. The restriction enzymes EcoR I, Nco I, Sac I, Fse I, and Hind III (New England Biolabs, Ipswich, MA) were used for cleavage. A positive control sample for hybridization was obtained by combining plasmid DNA, pDAS1740 (pDAB3812), with genomic DNA from a conventional control in a ratio of approximately equivalent to 1 copy of the transgene to the maize genome, and digested using the same methods and restriction enzyme as for the test samples . DNA from conventional control corn (XHH13) was digested using the same restriction enzyme methods and methods as for the test samples to be used as a negative control.

The digested DNA samples were precipitated using Quick-Precip (Edge BioSystems, Gaithersburg, MD) and resuspended in 1x Blue Juice (Invitrogen, Carlsbad, CA), adjusted to the volume required for loading onto the gel. DNA samples and molecular size markers were then electrophoresed on a 0.8% agarose gel using 1x TBE buffer (Fisher Scientific, Pittsburgh, PA) at 55-65 volts for approximately 18-22 hours to achieve fragment separation. The gels were stained with ethidium bromide (Invitrogen, Carlsbad, CA) and the DNA was visualized in ultraviolet (UV) light.

Example 2.3 Southern transfer and membrane processing

Southern blot analysis was performed essentially as described by Memelink et al. (1994) Southern, Northern, and Western Blot Analysis. Plant Mol. Biol. Manual F1: 1-23. Briefly, after electrophoretic separation and visualization of DNA fragments, the gels were depurinized using 0.25 N HCl (Fisher Scientific, Pittsburgh, PA) for about 15 minutes and then exposed to a denaturing solution (AccuGENE, Sigma, St. Louis, MO) for about 30 minutes followed by a neutralizing solution (AccuGENE, Sigma, St. Louis , MO) for at least 30 minutes. Southern transfer was performed overnight onto nylon membranes (Roche Diagnostics, Indianapolis, IN) using a 10 × SSC capillary system (Sigma, St. Louis, MO). After transfer, the membranes were washed in a 2x SSC solution and DNA was coupled to the membrane by cross-linking under the influence of UV. This method resulted in Southern blot membranes ready for hybridization.

Example 2.4 Labeling and hybridization of DNA probes

DNA fragments associated with the nylon membrane were detected using a labeled probe. The probes used for the study were created using PCR-based incorporation of digoxigenin-labeled (DIG) nucleotide, [DIG-11] -dUTP, from fragments formed by primers specific for gene elements and other regions of pDAS1740 plasmid. The creation of DNA probes by PCR synthesis was carried out using a kit for the synthesis of DIG probes in PCR (Roche Diagnostics, Indianapolis, IN), following the methods recommended by the manufacturer. The list of probes used for the study is shown in table 1.

Labeled probes were analyzed using agarose gel electrophoresis to determine their quality and quantity. The required amount of labeled probe was then used for hybridization with the target DNA on nylon membranes to detect specific fragments by the methods described for DIG Easy Hyb solution (Roche Diagnostics, Indianapolis, IN). Briefly, blots on nylon membranes with DNA fixed on them were briefly washed in 2 × SSC and pre-hybridized with 20-25 ml of pre-heated DIG Easy Hyb solution in hybridization vessels at approximately 50 ° C for a minimum of 30 minutes in a hybridization furnace. Then the pre-hybridization solution was drained and replaced with 20 ml of the pre-heated DIG Easy Hyb solution containing the required number of specific probes, previously denatured by boiling in water for 5 minutes. Then, a hybridization step was carried out at about 40-60 ° C. overnight in a hybridization furnace.

Example 2.5 Detection

At the end of hybridization with probes, DIG Easy Hyb solutions containing probes were poured into clean tubes and stored at -20 ° C. Such probes can be reused 2-3 times according to the method recommended by the manufacturer. Membrane blots were washed briefly and then washed twice in clean plastic containers using a low hardness wash buffer (2 × SSC, 0.1% SDS) for about 5 minutes at room temperature, then washed twice with a high hardness wash buffer (0 , 1 × SSC, 0.1% SDS), each time for 15 minutes at about 65 ° C. The membrane blots were then transferred to other clean plastic containers and washed briefly with 1x washing buffer from a set of DIG washing and blocking buffers (Roche Diagnostics, Indianapolis, IN) for about 2 minutes, then blocked in 1 x blocking buffer for a minimum of 30 minutes followed by incubation with anti-DIG-AP antibody (alkaline phosphatase) (1: 5000 dilution, Roche Diagnostics, Indianapolis, IN) in 1x blocking buffer for a minimum of 30 minutes. After 2-3 washes with 1x wash buffer, specific DNA probes that remained bound to the membrane blots and DIG-labeled DNA standards were visualized using a CDP-Star chemiluminescence nucleic acid detection system (Roche Diagnostics, Indianapolis, IN), following manufacturer's recommendations. Blots were exposed with a chemiluminescence recording film (Roche Diagnostics, Indianapolis, IN) for one or more time periods (time points) to determine hybridizing fragments and visualize molecular size standards. Films were then developed using an All-Pro 100 Plus developer (Konica SRX-101), and images were scanned to report data. The number and sizes of detected bands were documented for each probe. The DIG-labeled molecular weight marker II (MWM DIG II), visible after the detection of DIG, as described, was used to determine the sizes of the hybridizing fragments on Southern blots.

Example 2.6. Probe removal

DNA probes were removed from the membrane blots after acquiring Southern hybridization data, and the membrane blots could be reused for hybridization with another DNA probe using methods recommended by the manufacturer (DIG Application Manual for Filter Hybridization (2003). Roche Diagnostics). Briefly, after signal detection and exposure with film, the membrane blots were thoroughly washed with Milli-Q water and then washed twice in probe removal buffer (0.2 N NaOH, 0.1% SDS) for about 15 minutes at room temperature or at 37 ° C. Then the membrane blots were briefly washed in 2 × SSC, after which they were ready for pre-hybridization and hybridization with another DNA probe. Membrane blots were exposed with a new chemiluminescence recording film to ensure that all DNA probes were removed before proceeding with the next hybridization. After re-exposure, the films were stored together with a packet of data on the previous hybridization in the study file for the report.

Example 2.7 Southern Blot Results

The expected and observed fragment sizes in the case of a specific cleavage and probe based on sites for the known restriction enzymes pDAS 1740 / Fsp I fragment are shown in Table 2. As a result of these cleavages and hybridization, two types of fragments were identified: internal fragments, in which known restriction sites flanked the probe region and which were completely within the pDAS 1740 / Fsp I fragment, and border fragments, in which case the site for the known enzyme is located at one end of the probe region, and the second site is supposedly localized in the genome of maize. The sizes of the boundary fragments varied depending on the event, since in most cases the integration sites of DNA fragments are unique for each event. Border fragments provide a means for localizing the restriction enzyme site relative to integrated DNA and for estimating the number of DNA insertions. Based on Southern blot analyzes performed in this study, it was concluded that a single copy of the intact PTU aad-1 from plasmid pDAS 1740 / Fsp I was inserted into the maize genome in the event of DAS-40278-9, as detailed on the insertion map (figures 2, 3).

table 2 Predicted and Observed Hybridizing Fragments in Southern Blot Analysis DNA probe Restriction enzymes Expected fragment sizes (bp) ¹ Observed fragment sizes (bp) ² aad-1 EcoR I pDAS1740 8512 8512 XHH13 no no DAS-40278-9 > 3382 (border) ~ 12000 Nco i pDAS1740 8512 8512 XHH13 no no DAS-40278-9 > 2764 (border) ~ 4000 Sac i pDAS1740 8512 8512 XHH13 no no DAS-40278-9 > 4389 (border) ~ 16000 Fse i / hind iii pDAS1740 3361 3361 XHH13 no no DAS-40278-9 3361 3361 ZmUbi1 promoter Nco i pDAS1740 8512 8512, ~ 3600 * XHH13 no ~ 3600 * DAS-40278-9 > 3472 (border) ~ 6300, ~ 3600 * Sac i pDAS1740 8512 8512, ~ 3800 * XHH13 no ~ 3800 * DAS-40278-9 > 4389 (border) ~ 3800 *, ~ 16000 Fse i / hind iii pDAS1740 3361 3361, ~ 6400 * XHH13 no ~ 6400 * DAS-40278-9 3361 3361, ~ 6400 * # Terminator ZmPer5 Nco i pDAS1740 8512 8512, ~ 3900 * XHH13 no ~ 3900 * DAS-40278-9 > 2764 (border) ~ 4000, ~ 3900 * Sac i pDAS1740 8512 8512, ~ 9000 * XHH13 no ~ 9000 * DAS-40278-9 > 1847 (border) ~ 1900, ~ 9000 * Fse i / hind iii pDAS1740 3361 3361, ~ 2100 * XHH13 no ~ 2100 * DAS-40278-9 3361 3361, ~ 2100 * RB7 mar4 Nco i pDAS1740 8512 8512 XHH13 no no DAS-40278-9 > 2764 (border)> 3472 (border) ~ 4000
~ 6300 Sac i pDAS1740 8512 8512 XHH13 no no DAS-40278-9 > 1847 (border)> 4389 (border) ~ 1900
~ 16000 RB7 mar3 Nco i pDAS1740 8512 8512 XHH13 no no DAS-40278-9 > 2764 (border)> 3472 (border) ~ 4000
~ 6300 Sac i pDAS1740 8512 8512 XHH13 no no DAS-40278-9 > 1847 (border)> 4389 (border) ~ 1900
~ 16000 Wreck Nco i pDAS1740 8512 8512 XHH13 no no DAS-40278-9 no no Sac i pDAS1740 8512 8512 XHH13 no no DAS-40278-9 no no Note: * An asterisk after the observed fragment size indicates the hybridization of the endogenous sequence that was detected in all samples (including negative controls).
# Same values in normal control, BC3S1, and in some BC3S2 samples.
^{1 The} expected fragment sizes are based on the map of plasmid pDAS 1740 (pDAB3812), which is shown in figure 1.
^{2 The} observed fragment sizes were estimated approximately based on such assays, and they are based on the indicated fragment sizes of the DIG-labeled DNA molecular weight marker II. Due to the inclusion of DIG molecules for visualization, marker fragments in the experiment were usually 5-10% more than their actual molecular weight.

Restriction enzymes having a unique restriction site in plasmid pDAS1740, EcoR I, Nco I, Sac I, Fse I / Hind III were selected to characterize the aad-1 gene insert in the event of DAS-40278-9. It was assumed that the boundary fragment with a length of> 3382 bp,> 2764 bp,> 4389 bp hybridizes with the probe of the aad-1 gene after cleavage of EcoR I, Nco I and Sac I, respectively (table 2). One aad-1 hybridization band was observed with a length of ~ 12000 bp, ~ 4000 bp and ~ 16000 bp when using EcoR I, Nco I, and Sac I, respectively, which indicates the presence of a single aad-1 insertion site in the maize genome in the event of DAS-40278-9. Double cleavage using Fse I and Hind III was selected to release a 3361 bp fragment that contains the aad-1 plant transcriptional unit (PTU, promoter / gene / terminator) (Table 2). Predicted 3361 bp fragment observed using a probe for the aad-1 gene after Fse I / Hind III digestion. The results obtained by digestion with all four enzymes / enzyme combination of sample DAS-40278-9, followed by hybridization with aad-1 probe, showed that one copy of the aad-1 intact PTU from plasmid pDAS1740 was inserted into the maize genome in case of a DAS event -40278-9.

The restriction enzymes Nco I, Sac I and Fse I / Hind III were selected to characterize the promoter region (ZmUbi1) for aad-1 in the event of DAS-40278-9. The Nco I and Sac I cleavages presumably form fragments of the boundary region with a length of> 3472 bp and> 4389 bp, respectively, observed upon hybridization with DNA probes specific for the ZmUbi1 promoter region (Table 2). Two hybridization bands ~ 6300 bp and ~ 3600 bp identified using a probe to the ZmUbi1 promoter after cleavage of Nco I. However, the band is ~ 3600 bp was present on all tracks with samples, including the usual controls, which indicates that the strip is ~ 3600 bp in length. is a band of a non-specific signal resulting from homologous binding of the probe to the ubiquitin promoter (ZmUbi1) obtained from maize with the endogenous maize ubi gene. On the contrary, the signal bandwidth is ~ 6300 bp detected in the tested samples DAS-40278-9, but not detected in the usual controls, which indicates that the band is ~ 6300 bp is specific for the probe probe ZmUbi1 from plasmid pDAS1740 and, therefore, is the expected Nco I / ZmUbi1 band shown in Table 2. Two hybridization bands of ~ 3800 bp were identified in a similar manner. and ~ 16000 bp using the probe of the ZmUbi1 promoter after cleavage of Sac I. A band of ~ 3800 bp were detected on all tracks with samples, including the usual controls, and therefore, it was believed that it was the result of nonspecific hybridization of the probe to the ZmUbi1 promoter with the endogenous corn ubi gene. The hybridization band of ~ 16000 bp, which is present only in samples DAS-40278-9, is regarded as the expected band of Sac I / ZmUbi1. Double cleavage using Fse I / Hind III was supposed to release a 3361 bp PTU aad-1 fragment that hybridizes with the probe of the ZmUbi1 promoter (Table 2). Such a band is 3361 bp and a nonspecific hybridization band of ~ 6400 bp in length identified using a probe to the ZmUbi1 promoter after Fse I / Hind III digestion. ~ 6400 bp long consider the result of nonspecific binding of the probe to the ZmUbi1 promoter with the endogenous ubi gene of maize, since such a band is present on all tracks with samples, including the usual controls. In addition, another band was observed, very close to the ~ 6400 bp band, in the normal control, BC3S1, and in some of the BC3S2 samples. This additional band, very close to ~ 6400 bp, is also considered nonspecific, since it is present on tracks with the usual control samples XHH13 and is most likely associated with the genetic background XHH13.

The same restriction enzymes / combination of enzymes, Nco I, Sac I and Fse I / Hind III, was chosen to characterize the terminator region (ZmPer5) for aad-1 in the event of DAS-40278-9. It is believed that Nco I cleavage forms a fragment of the border region with a length of> 2764 bp detected by hybridization with DNA probes specific for the ZmPer5 terminator region (Table 2). Two hybridization bands ~ 4000 bp and ~ 3900 bp identified using a probe to the ZmPer5 terminator after cleavage of Nco I. Band ~ 3900 bp was present on all tracks with samples, including the usual controls, which indicates that the strip is ~ 3900 bp in length. is a non-specific signal band, probably due to the homologous binding of the probe to the peroxidase gene terminator derived from maize (ZmPer5) with the endogenous per maize gene. In contrast, the signal band is ~ 4000 bp detected in the tested samples DAS-40278-9, but not detected in the usual controls, which indicates that the strip length ~ 4000 bp is specific for the probe to the ZmPer5 terminator from plasmid pDAS 1740, and therefore it is the expected Nco I / ZmPer5 band indicated in Table 2. It was assumed that the boundary fragment is> 1847 bp in length. hybridizes with a probe to the ZmPer5 terminator after Sac I cleavage. Two hybridization bands ~ 1900 bp in length and ~ 9000 bp were detected using a probe to the ZmPer5 terminator after cleavage of Sac I. A band of ~ 9000 bp in length were detected on all paths with samples, including the usual controls, and therefore were regarded as non-specific hybridization of the probe to the ZmPer5 terminator with the endogenous per corn gene. The ~ 1900 bp hybridization band, which is present only in DAS-40278-9 samples, is regarded as the expected Sac I / ZmPer5 band. Double cleavage using Fse I / Hind III presumably releases a 3361 bp PTU fragment of aad-1 that hybridizes with the probe to the ZmPer5 terminator (Table 2). Such a band is 3361 bp and an additional non-specific hybridization band of ~ 2100 bp in length identified using a probe to the ZmPer5 terminator after Fse I / Hind III digestion. Additional lane ~ 2100 bp is the result of nonspecific binding of the probe to the ZmPer5 terminator with the endogenous gene of maize, since such a band is present on all tracks with samples, including negative controls. The results obtained using such cleavages of sample DAS-40278-9, followed by hybridization with probes to the ZmUbi1 promoter and ZmPer5 terminator, further confirmed that one copy of the intact PTU aad-1 from plasmid pDAS1740 was inserted into the maize genome in the event of the DAS-40278- event 9.

Restriction enzymes, Nco I and Sac I, were selected to characterize the remaining components from the pDAS1740 / Fsp I fragment in the case of the AAD-1 DAS-40278-9 event of maize (table 2). The DNA sequences of the components of RB7 Mar v3 and RB7 Mar v4 are more than 99.7% identical, so it was expected that DNA probes specific for RB7 Mar v3 or RB7 Mar v4 would hybridize with DNA fragments containing any of the RB7 Mar variants. It was assumed that two boundary fragments with a length of> 2764 bp and> 3472 bp hybridize with probes for RB7 Mar v4 and RB7 Mar v3 after Nco I digestion (Table 2). Two hybridization bands ~ 4000 bp and ~ 6300 bp observed using a probe to either RB7 Mar v4 or RB7 Mar v3 after Nco I digestion in samples DAS-40278-9. Similarly, two boundary fragments with a length of> 1847 bp were suggested. and> 4389 bp in the case of using probes RB7 Mar v4 and RB7 Mar v3 after splitting Sac I (table 2). ~ 1900 bp hybridization bands and ~ 16000 bp detected in samples DAS-40278-9 using an RB7 Mar v4 or RB7 Mar v3 probe after Sac I digestion

Taken together, the results of Southern hybridization obtained using such probes to the elements show that the DNA embedded in the case of the DAS-40278-9 event of maize contains intact PTU aad-1 along with the attachment sites to the RB7 Mar v3 and RB7 Mar v4 matrix at the 5'- and 3'-ends of the insert, respectively.

Example 2.8. Lack of core sequences

The combination of three DNA fragments in an equimolar ratio (table 1), covering the almost complete Fsp I region of the backbone (4867-7143 bp in plasmid pDAS1740) of plasmid pDAS1740, was used as a probe to the backbone to characterize the AAD-1 event in maize DAS-40278-9. To obtain the DAS-40278-9 event, the plasmid pDAS 1740 / Fsp I fragment was used; therefore, specific hybridization with a combination of probes to the backbone (Table 2) after digestion with any restriction enzyme was not expected. It was confirmed that a specific hybridization signal was not detected when using the probe to the core after cleavage of Nco I or Sac I in any of the samples DAS-40278-9. The positive control tracks contained the expected hybridizing bands, which suggests that the probes are hybridized with any homologous DNA fragments, if present in the samples. The data indicate that the insertion in the case of the DAS-40278-9 event of maize does not contain any sequence of the vector backbone outside the Fsp I region from plasmid pDAS1740.

Leaf samples from five different generations for event DAS-40278-9 were used for Southern blot analysis for molecular characterization. The integration pattern was investigated using digestion of the selected restriction enzyme and combination of probes to characterize the inserted gene, aad-1, as well as non-coding regions, including the promoter, gene expression terminator and matrix binding sites.

Southern blot characterization of the DNA embedded in the event of DAS-40278-9 indicates that a single intact copy of PTU aad-1 was integrated in the event of DAS-40278-9. The molecular masses shown using Southern hybridization in the case of a combination of a restriction enzyme and a probe were unique to the event and formed patterns for its identification. Hybridization patterns are identical for all five generations, suggesting that the insert is stable in the maize genome. Hybridization with probes spanning the region of the backbone outside the pDAS 1740 / Fsp I fragment for transformation from plasmid pDAS 1740 confirms that the sequences of the vector backbone were not included in event DAS-40278-9.

Example 3. Cloning and characterization of the DNA sequence of the insert and flanking border regions in the case of the event DAS-40278-9 corn

In order to characterize the inserted DNA and to describe the genomic insertion site, the DNA sequences of the insert and the border regions were determined in the event of DAS-40278-9. Everything was confirmed 8557 bp genomic sequence in the case of the event DAS-40278-9 containing 1873 bp 5’-flanking border sequence, 1868 bp 3’-flanking border sequence and 4816 bp DNA inserts. 4816 bp DNA insert contains an intact aad-1 expression cassette, incomplete MAR v3 259 bp long at the 5’-end and incomplete 1096 bp MAR v4 at the 3’s end. Sequence analysis revealed an insertion of 21 bp at the 5’ site of the compound upon integration and deletion of two pairs of nucleotides from the insertion locus of the maize genome. An insertion of one nucleotide pair was detected at the 3’ site of the compound upon integration between the maize genome and the DAS-40278-9 insert. A change in one base (T to C) was also detected in the insert at position 5212 in the non-coding region of the 3’-UTR. None of these changes affected the composition of the open reading frame of the aad-1 expression cassette.

PCR amplification based on the insert sequences and the border sequences of the DAS-40278-9 event confirmed that the border regions were from corn and that the connection regions could be used for event-specific identification of DAS-40278-9. The analysis of the sequence covering the regions of the compound showed that no new open reading frames (ORF> = 200 codons) occurred as a result of DNA insertion in the event of the DAS-40278-9 event, and also none of the genomic open reading frames were interrupted during integration DAS-40278-9 in the native genome of maize. In general, the characterization of the insert sequences and the border sequences of the AAD-1 maize event DAS-40278-9 showed that only one intact copy of the aad-1 expression cassette was integrated into the native maize genome.

Example 3.1 Extraction and quantification of genomic DNA

Genomic DNA was extracted from lyophilized or freshly prepared ground leaf tissues using a modified CTAB method. DNA samples were dissolved in 1 × TE (10 mM Tris, pH 8.0, 1 mM EDTA) (Fluka, Sigma, St. Louis, MO) and quantified using the Pico Green method according to the manufacturer's instructions (Molecular Probes, Eugene, OR). For PCR analysis, DNA samples were diluted with special purification water for molecular biology (5 PRIME, Gaithersburg, MD) to obtain a concentration of 10-100 ng / μl.

Example 3.2. PCR primers

Table 3 shows a list of primer sequences that were used to clone the DNA insert and the flanking border regions of the DAS-40278-9 event, the positions and descriptions being shown in Figure 4. Table 4 shows a list of primer sequences that were used to confirm the sequence of the insert and border sequences. The positions of the primer are marked in figures 4 and 5, respectively. All primers were synthesized at Integrated DNA Technologies, Inc. (Coralville, IA). Primers were dissolved in water (5 PRIME, Gaithersburg, MD) to a concentration of 100 μM to obtain a mother liquor and diluted with water to a concentration of 10 μM to obtain a working solution.

Example 3.3 Walk through the genome

A universal genome walking kit (Clontech Laboratories, Inc., Mountain View, CA) was used to clone the 5'- and 3'-flanking border sequences of the maize DAS-40278-9 event. According to the manufacturer's instructions, approximately 2.5 μg of genomic DNA AAD-1 events of maize DAS-40278-9 were digested overnight using EcoR V, Stu I (both enzymes are supplied) or Sca I (New England Biolabs, Ipswich, MA) . Digested DNA is purified using ^{DNA Clean and Concentrator TM -25 (ZYMO} Research, Orange, CA), followed by ligation with adapters Genome Walker ^TM, to construct the Genome Walker ^TM library. Genome Walker ^TM each library was used as template DNA for the primary PCR amplification with adapter primer AP1, supplied in the set, and each specific for primer design and 5End3812_A 3End3812_C. Then, one microliter after 1:25 dilution of the mixture for the primary PCR reaction was used as a matrix for secondary PCR amplification with the AP2 female adapter primer and each 5End3812_B and 3End3812_D primer-specific design. TaKaRa LA Taq ^™ HS (Takara Bio Inc., Shiga, Japan) was used in PCR amplification. 50 μl of the PCR reaction mixture used 1 μl of the DNA template, 8 μl of 2.5 mM dNTP mixture, 0.2 μM of each primer, 2.5 units of TaKaRa LA Taq ^TM HS DNA polymerase, 5 μl of 10 x PCR buffer LA II (plus Mg2 +) and 1.5 μl of 25 mM MgCl ₂ . Specific PCR conditions are shown in table 5.

Example 3.4. Traditional PCR

Standard PCR was used to clone and confirm the DNA insert and the border sequence in case of the DAS-40278-9 event of maize. DNA polymerase ^{TaKaRa LA Taq TM (Takara Bio Inc.} , Shiga, Japan), HotStarTaq (Qiagen, Valencia, CA), a system for enhanced precision PCR (Roche Diagnostics, Inc., Indianapolis, IN) or a mixture of High-Fidelity PCR Cloning Enzyme and Master Mix Easy-A® (Stratagene, LaJolla, CA) were used for routine PCR amplification according to methods recommended by the manufacturer. Specific PCR conditions and amplicon descriptions are given in Table 6A.

Example 3.5 Detection of a PCR product, purification, subcloning of PCR products and sequencing

PCR products were examined by electrophoresis using a 1.2% or 2% E-gel (Invitrogen, Carlsbad, CA) according to the instructions for the product. Fragment size was estimated by comparison with DNA markers. If necessary, PCR fragments were purified by excising fragments from a 1% agarose gel in 1x TBE stained with ethidium bromide using a QIAquick gel extraction kit (Qiagen, Carlsbad, Calif.).

PCR fragments were subcloned into pCR®4-TOPO® using the TOPO TA Cloning® Sequencing Kit (Invitrogen, Carlsbad, CA) according to the product instructions. In particular, two to five microliters of TOPO® cloning reaction mixture was used to transform chemically competent One Shot TOP 10 cells following the manufacturer's instructions. Cloned fragments were confirmed by plasmid DNA miniproducts (QIAprep Spin Miniprep kit, Qiagen, Carlsbad, CA) followed by restriction digestion of EcoR I or direct PCR on colonies using T3 and T7 primers supplied in the kit. Then plasmid DNA or stock solutions in glycerol of the selected colonies was sent for sequencing.

After subcloning, the putative target PCR products were first sequenced to confirm that the expected DNA fragments were cloned. Colonies containing the corresponding DNA sequences were selected for primer-mediated walks to determine complete DNA sequences. Sequencing was performed in Cogenics (Houston, TX).

The final assembly of the insert and boundary sequences was carried out using a Sequencher computer program (version 4.8, Gene Codes Corporation, Ann Arbor, MI). Annotation of the insert and border sequences of the DAS-40278-9 event of corn was prepared using Vector NTI (versions 10 and 11, Invitrogen, Carlsbad, CA).

Homology searches were performed using the BLAST program and the GenBank database. Open reading frame analysis (ORF) using Vector NTI (version 11, Invitrogen) was performed to identify ORFs (> = 200 codons) in complete insert and flanking border sequences.

Example 3.6 5’-end border sequence

A DNA fragment was amplified from each library obtained by walking through the genome in the case of the DAS-40278-9 event in maize using a set of specific nested primers for the 5 ′ end of the transgene. A PCR product of approximately 800 bp in length observed in both libraries obtained by walking through the genome in the case of event DAS-40278-9 using EcoR V and Stu I. The library obtained by walking through the genome using Sca I gave a product of about 2 kb. Fragments were cloned into pCR®4-TOPO® and six colonies were randomly selected from each library for final sequencing to confirm that the insert contained the expected sequences. Complete sequencing of primer-mediated inserts revealed that fragments amplified from libraries obtained by genomic walking using Stu I, EcoR V, and Sca I in the case of DAS-40278-9 in maize were 793, 822, and 2132 long bp, respectively. DNA fragments formed from libraries obtained as a result of a genome walk using Stu I and EcoR V were 100% identical to the DNA fragment generated from a library obtained as a result of a genome walk using Sca I, which indicates such DNA fragments are amplified from the 5 ′ region of the transgene insert. BLAST search of the resulting 1873 bp maize genomic sequence of maize showed high similarity to the sequence of the corn BAC clone. In addition, sequence analysis of the insertion compound showed that the sequence was 917 bp in length. MAR v3 in the 5’-end region is shortened compared to the plasmid pDAS1740 / Fsp I fragment, with 259 bp remaining incomplete MAR v3 in the 5’ region of the aad-1 expression cassette.

Example 3.7. Boundary sequence at the 3’ end

A DNA fragment of approximately 3 kbp amplified from the library obtained by walking through the genome using Stu I in the case of the DAS-40278-9 event of maize using a set of nesting primers specific for the 3'-end of the transgene. The DNA fragment was cloned into pCR ^(R) 4-TOPO® and ten colonies were randomly selected for final sequencing to confirm the insertion of the expected sequences. Three clones with the expected inserts were completely sequenced to obtain a 2997 bp DNA fragment. Sequence analysis of such a DNA fragment revealed an incomplete MAR v4 element (70 bp of its 5'-region was missing) and a 1867 bp corn genomic sequence A search using BLAST showed that the genomic DNA sequence was 1867 bp in length. 100% coincided with the sequence in the same maize BAC clone that was identified using the 5'-boundary sequence.

Example 3.8. DNA Insertion Sequences and Compounds

The DNA insert and regions of the compound were cloned for the case of the DAS-40278-9 event of maize using the PCR based methods described previously. Five primer pairs were constructed based on 5'- and 3'-flanking border sequences and the expected transgene sequence. A total of five overlapping DNA fragments were cloned and sequenced (amplicon 1, 1767 bp long, amplicon 2, 1703 bp long, amplicon 3, 1700 bp long, amplicon 4, 1984 bp, and amplicon 5, 1484 long) bp) (figure 4). Complete insertion sequences and flanking boundaries were assembled based on the overlapping sequence of five fragments. The final sequence confirms the presence of 4816 bp insert DNA obtained from pDAS 1740 / Fsp I, 1873 bp 5’-flanking border sequence and 1868 bp 3’s flanking border sequence. 4816 bp DNA insert contains an intact aad-1 expression cassette, 259 bp incomplete MAR v3 at the 5’s end and 1096 bp incomplete MAR v4 at the 3’-end (SEQ ID NO: 29).

At least two clones for each pair of primers were used for primer-mediated walks to obtain complete information about the DNA insert sequence and its border sequences. Sequence analysis revealed an insertion of 21 bp in the region of the connection on the 5’ side of the integration site between genomic DNA and integrated incomplete MAR v3 from pDAS1740 / Fsp I. BLAST search results and Vector NTI analysis showed that the DNA insert was 21 bp long. does not show homology with either the DNA of any plant species or the plasmid DNA pDAS1740. The insertion of one nucleotide pair was detected in the region of the connection on the 3’ side of the integration site between maize genomic DNA and incomplete MAR v4 from pDAS1740 / Fsp I. DNA integration also led to the deletion of two nucleotides at the insertion locus of the maize genome (Figure 6). In addition, the difference in one nucleotide (T to C) at position 5212 bp observed in the untranslated 3’-UTR region of the DNA insert (SEQ ID NO: 29). However, none of these changes, apparently, is important for the expression of aad-1 or the creation of any new ORF (> = 200 codons) throughout the connection areas of the insert DAS-40278-9.

Example 3.9. Confirmation of the genomic sequences of corn

In order to confirm the transgene insertion site in the case of the DAS-40278-9 event in the maize genome, PCR amplification was performed with different pairs of primers (Figure 4). Genomic DNA corresponding to event DAS-40278-9, and the genomic DNA of other transgenic or non-transgenic maize lines were used as a template. Two aad-1 specific primers, AI5End01 and AI5End02, and two primers designed according to the 5'-terminal boundary sequence, 1F5End01 and 1F5End02, were used to amplify the DNA fragment spanning the region of the aad-1 gene to the 5'-terminal boundary sequence. Similarly, to amplify a DNA fragment spanning the region of the aad-1 gene to a 3 ′ terminal boundary sequence, a 1F3End05 primer derived from the 3 ′ terminal boundary sequence and aad-1 specific primer AI3End01 were used. DNA fragments of the expected sizes were amplified only with genomic DNA in the case of the AAD-1 DAS-40278-9 event, each pair of primers consisting of one primer located at the flanking border of the AAD-1 DAS-40278-9 event of corn and one specific for aad-1 primer. Control DNA samples did not give PCR products with the same primer pairs, indicating that the cloned 5’ and 3 ′ terminal boundary sequences are indeed sequences overlying and underlying from the aad-1 gene’s built-in design, respectively. It should be noted that a weak band of approximately 8 kbp observed in all maize samples, including the DAS-40278-9 maize AAD-1 event, the DAS-40474 maize AAD-1 event, and the XHH13 non-transgenic maize line when a pair of primers 1F5End01 and AI5End01 were used for PCR amplification. The observed weak band (on the obtained gel) could be the result of nonspecific amplification in the maize genome using such a pair of primers.

To further confirm the DNA insertion in the maize genome, two primers located at the 5'-terminal boundary sequence, 1F5End03 and 1F5End04, and two primers located at the 3'-terminal boundary sequence, 1F3End03 and 1F3End04 were used to amplify DNA fragments spanning insertion locus. PCR amplification with either a pair of primers 1F5End03 / 1F3End03 or a pair of primers 1F5End04 / 1F3End04 resulted in a fragment of the expected size of approximately 8 kb. from genomic DNA in the case of the AAD-1 event of maize DAS-40278-9. In contrast, no PCR products with genomic DNA were obtained in the case of the DAD-40474-7 maize AAD-1 event or in the case of the non-transgenic maize line XHH13. Considering the fact that the AAD-1 event of corn DAS-40278-9 and the event DAS-40474-7 are the result of HiII transformation followed by backcrossing of the original lines with transgenic events with the corn line XHH13, most of the genome in the case of each of these two events theoretically comes from the XHH13 corn line. It is very likely that only flanking boundary sequences near the aad-1 transgene were transferred from the original genomic DNA and preserved during the introgression of the AAD-1 event, while other regions of the genomic sequences could be replaced by XHH13 genomic sequences. Thus, it is not surprising that not a single fragment was amplified with genomic DNA in the case of the AAD-1 event of maize DAS-40474-7 and XHH13 using either a pair of primers 1F5End03 / 1F3End03 or a pair of primers 1F5End04 / 1F3End04. Fragments approximately 3.1 and 3.3 kb in length amplified using a pair of primers 1F5End03 / 1F3End03 and 1F5End04 / 1F3End04, respectively, with the genomic DNA of the HiII and B73 maize lines, but not the A188 maize line. The results show that the border sequences originated from the genome of the B73 maize line.

Additional cloning of the genomic DNA of maize from B73 / HiII was performed to ensure the reliability of the flanking border sequences. PCR amplified fragments were sequenced to confirm data on the DNA insertion region integrated at a particular position of the B73 / HiII genomic DNA. Primers were designed based on the obtained sequence. The Amp 1F / Amp 5R primer set was used to amplify a 2212 bp fragment covering the region of 5'- to 3'-compounds from the native B73 / HiII genome without DNA insertion. Sequence analysis showed that there is a deletion of two pairs of nucleotides from the native B73 genome at the transgene insertion locus. Analysis of DNA sequences from the cloned fragment of the native B73 genome revealed one ORF (> = 200 codons) located below the 3’ region of the compound upon integration. In addition, there are no other ORFs along the entire length of the original locus into which the integration with the AAD-1 event of maize DAS-40278-9 occurred. A BLAST search also confirmed that both the 5-terminal and 3-terminal boundary sequences available for the DAS40278-9 event are located nearby in the same maize BAC clone.

Given the uniqueness of the 5’-compound region when integrating the DAD-40278-9 maize AAD-1 event, two pairs of specific PCR primers, 1F5EndT1F / 1F5EndT1R and Corn278-F / Corn278-R, were designed to amplify such a compound of the insert from the genomic plant. As expected, the desired DNA fragment was formed only in genomic DNA in the case of the AAD-1 event of maize DAS-40278-9, but not in other transgenic or non-transgenic maize lines. Thus, these two pairs of primers can be used as identifiers specific to the AAD-1 event of maize DAS-40278-9.

Example 4. Characterization of the genome using flanking SSR markers DAS-40278-9

In order to characterize and describe the genomic insertion site, marker sequences localized near the insert were determined. A panel of polymorphic SSR markers was used to identify and map the position of the transgene. Event pDAS 1740-278 is located on chromosome 2 at about 20 cM between the SSR markers UMC1265 and MMC0111 at about 20 cM on the 2008 DAS maize linkage map. Table 6 summarizes the primer information for these two markers that were found in close proximity to the pDAS1740-278 transgene.

Example 4.1 Isolation of gDNA

gDNA was extracted from leaf-cut tissue samples using a DNEasy 96 plant test kit (Qiagen, Valencia, California). Modifications to the protocol for automation have been implemented. Isolated gDNA was quantified using PicoGreen® dye from Molecular Probes, Inc. (Eugene, OR). The concentration of gDNA was adjusted to 5 ng / μl by dilution of all samples using sterile deionized water.

Example 4.2 GDNA screening using markers

The diluted gDNA was genotyped using a subgroup of simple repeat sequence markers (SSRs). SSR markers were synthesized in Applied Biosystems (Foster City, California) using direct primers labeled with 6-FAM, HEX / VIC or NED fluorescent labels (cyan, green, and yellow, respectively). Markers were divided into groups or panels based on their fluorescent label and amplicon size to facilitate multiplexing and analysis after PCR.

PCR was performed in 384-well assay plates, with each reaction mixture containing 5 ng of genomic DNA, 1.25X PCR buffer (Qiagen, Valencia, California), 0.20 μM of each forward and reverse primer, 1.25 mM MgCl _2, 0.015 mM of each dNTP and 0.3 units of DNA polymerase HotStart Taq (Qiagen, Valencia, California ). Amplification was performed in a GeneAmp 9700 PCR system with a dual 384-well module (Applied Biosystems, Foster City, California). The following amplification program was used: (1) initial Taq activation at 95 ° C for 12 minutes; (2) 30 seconds at 94 ° C; (3) 30 seconds at 55 ° C; (4) 30 seconds at 72 ° C; (5) repeating steps 2-4 over 40 cycles; and (6) 30 minute final extension at 72 ° C. The PCR products for each panel of SSR markers were multiplexed together by adding 2 μl of each PCR product of the same plant to sterile deionized water to a total volume of 60 μl. Of the multiplexed PCR products, 0.5 μl was added to 384-well loading plates containing 5 μl of loading buffer consisting of a mixture of 1: 100 standard size in pairs of GeneScan 500 LIZ nucleotides and ABI HiDi formamide (Applied Biosystems, Foster City , California). Samples were then loaded into an ABI Prism 3730x1 DNA analyzer (Applied Biosystems, Foster City, California) for capillary electrophoresis using manufacturer recommendations using a total working time of 36 minutes. Marker data was collected using an ABI Prism 3730x1 version 4.0 computer program for automated sequence data collection, and extracted using GeneMapper 4.0 computer program (Applied Biosystems) to characterize alleles and determine fragment sizes.

Example 4.3 SSR Marker Analysis Results

Primer data for flanking markers that are identified in close proximity to the transgene are shown in Table 6. The two most closely associated markers, UMC1265 and MMC0111, are located approximately 20 cM from the transgene insert in chromosome 2.

Example 5. Characterization of aad-1 protein in the case of the event DAS-40278-9

The biochemical properties of the recombinant aad-1 protein obtained from transgenic corn, in which the event DAS-40278-9 takes place, were characterized. Used polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE stained with Coomassie blue, and methods for detecting glycoproteins), Western blot, immunodiagnostic test strip assays, time-of-flight mass spectrometry with laser desorption / ionization using the TOI D matrix F (MAL MS) and protein sequencing analysis using tandem MS to characterize the biochemical properties of the protein.

Example 5.1. Immunodiagnostic Strip Analysis

The presence of aad-1 protein in the tissue of the DAS-40278-9 sheet was confirmed using commercially available immunodiagnostic test strips from American Bionostica. The strips made it possible to distinguish between transgenic and non-transgenic plants when testing untreated leaf extracts (data not shown). Nontransgenic extracts (XHH13) did not contain detectable amounts of immunoreactive protein. This result was also confirmed in a Western blot analysis.

To test the expression of aad-1 protein, immunodiagnostic strip assays were performed. Four leaves cut by punching a sample of the leaf were collected from each XHH13 plant (control plant) and plants with the DAS-40278-9 event, clamping the tissue with lids in the form of a snap-on cap of individual 1.5 ml labeled microcentrifuge tubes. After delivery to the laboratory, 0.5 ml of aad-1 extraction buffer (American Bionostica, Swedesboro, NJ) was added to each tube, and the tissue was homogenized using a disposable pestle, followed by shaking of the sample for ~ 10 seconds. After homogenization, a test strip was placed in a test tube and allowed to develop for ~ 5 minutes. The presence or absence of aad-1 protein in the plant extract was confirmed based on the appearance (or absence) of a test line on the immunodiagnostic strip. After the expression of aad-1 protein was confirmed for a transgenic event, corn stem tissue was collected, lyophilized and stored at about -80 ° C until use.

Example 5.2. Purification of aad-1 protein from corn

An immunologically purified aad-1 protein obtained from maize was prepared (molecular weight: ~ 33 kDa) or crude aqueous extracts from corn stalk tissue. All leaf and stem tissue was collected and transported to the laboratory as follows. Leaves were cut from the plant with scissors and placed in tissue bags and stored at about -20 ° C for future use. Separately, the stems were cut just above the soil level, placed in fabric bags and immediately frozen at about -80 ° C for ~ 6 hours. Then the stems were placed in a lyophilizer for 5 days to remove water. After the fabrics were completely dried, they were crushed to a fine powder with dry ice and stored at about -80 ° C until they were needed.

The aad-1 protein obtained from corn was extracted from lyophilized stem tissue in a phosphate-based buffer (see buffer components in Table 7), weighing ~ 30 grams of lyophilized tissue into a 1000 ml chilled glass shredder and adding 500 ml of extraction buffer. The tissue was pulverized from above for 60 seconds, and soluble proteins were collected by centrifuging the sample for 20 minutes at 30,000 × g. The precipitate was re-extracted as described, and the supernatants were combined and filtered through a 0.45 μm filter. Filtered supernatants were loaded at approximately + 4 ° C on an immunoaffinity anti-aad-1 column onto which a monoclonal antibody prepared by Strategic Biosolution Inc. was conjugated. (mAb 473F1 85.1; purified on protein A; batch No.: 609.03C-2-4; 6.5 mg / ml (total ~ 35.2 mg)) (Windham, ME) conjugated to CNBr-activated Sepharose 4B (GE Healthcare, Piscataway, NJ). Unbound proteins were collected and the column was washed thoroughly with pre-chilled buffer based on 20 mM ammonium bicarbonate, pH 8.0. Bound proteins were eluted with 3.5 M NaSCN (Sigma, St. Louis, MO), 50 mM Tris buffer (Sigma, St. Louis, MO), pH 8.0. Seven fractions of 5 ml were collected, and fractions numbered 2 → 7 were dialyzed overnight at approximately + 4 ° C against 10 mM Tris buffer, pH 8.0. Fractions were examined using SDS-PAGE and Western blot, and the remaining samples were stored at approximately + 4 ° C until use in subsequent analyzes.

Table 7 Commercially available reference substances used in this study Reference substance The product's name Batch number Analysis Link Soybean Trypsin Inhibitor GelCode Glycoprotein Staining Kit Component IA110577 Glycosylation Pierce, Catalog No. 1856274 Horseradish peroxidase GelCode Glycoprotein Staining Kit Component JG124509 Glycosylation Pierce, Catalog No. 1856273 Fraction V of bovine serum albumin (BSA) Set of standards for analysis of pre-diluted BSA protein FH71884A Glycosylation, SDS-PAGE and Western Blot Pierce, Catalog No. 23208 Pre-stained molecular weight markers Pre-stained Novex Sharp Protein Markers 469212 and 419493 Western blot Invitrogen, LC5800 catalog number, molecular weight markers 260, 160, 110, 80, 60, 50, 40, 30, 20, 15, 10 and 3.5 kDa Molecular weight markers Protein Marker Blend Invitrogen Mark 12 39983 and 399895 SDS PAGE Invitrogen, LC5677 catalog number, molecular weight markers 200, 116.3, 97.4, 66.3, 55.4, 36.5, 31.0, 21.5, 14.4, 6.0, 3, 5 and 2.5 kDa

The protein that binds to the immunoaffinity column was examined in SDS-PAGE, and the results showed that the eluted fractions contained aad-1 protein with an approximate molecular weight of 33 kDa. In addition, Western blotting was also performed, and the result was positive for aad-1 protein. The aad-1 protein obtained from maize was isolated from ~ 30 g of lyophilized stem material.

Example 5.3 SDS-PAGE and Western Blot

Lyophilized tissue from plant stems with event DAS-40278-9 and XHH13 (~ 100 mg) was weighed into 2 ml microcentrifuge tubes and extracted using ~ 1 ml PBST (Sigma, St. Louis, MO) containing 10% protease inhibitor mixture plants (Sigma, St. Louis, MO). Extraction was facilitated by adding 4 small ball bearings and grinding the sample using Geno-Grinder for 1 minute. After grinding, the samples were centrifuged for 5 minutes at 20,000 × g, and the supernatants were mixed 4: 1 with 5x Laemmli sample buffer (2% SDS, 50 mM Tris, pH 6.8, 0.2 mg / ml bromphenol blue, 50% (w / w) glycerol containing 10% freshly added 2-mercaptoethanol) and heated for 5 minutes at ~ 100 ° C. After brief centrifugation, 45 μl of the supernatant was directly loaded onto a BioRad Criterion SDS-PAGE gel (Bio-Rad, Hercules, CA) inserted into a Criterion Cell gel chamber. The positive reference standard obtained from microorganisms aad-1 was resuspended at a concentration of 1 mg / ml in PBST, pH 7.4, and then PBST was diluted. The sample was then mixed with Lammley Bio-Rad buffer with 5% 2-mercaptoethanol and processed as previously described. Electrophoresis was performed using a Tris / Glycine / SDS buffer (Bio-Rad, Hercules, CA) at a voltage of 150-200 V up to the moment when the dye front appeared at the end of the gel. After separation, the gel was cut in half, and one half was stained with blue Pierce GelCode protein dye, and the other half was electro-blotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA) using a Mini trans-blot electrophoretic transfer cell (Bio-Rad, Hercules) , CA) for 60 minutes at a constant voltage of 100 volts. The transfer buffer contained 20% methanol and Tris / glycine buffer from Bio-Rad. For immunodetection, the membrane was examined using an aad-1-specific polyclonal rabbit antibody (Strategic Biosolution Inc., Newark, DE, purified on Protein A rabbit polyclonal antibody, batch No.: DAS F1 197-15 1, 1.6 mg / ml). A goat antibody conjugate against rabbit IgG (H + L) and alkaline phosphatase (Pierce Chemical, Rockford, IL) was used as the second antibody. The SigmaFast BCIP / NBT substrate was used for the development and visualization of immunoreactive protein bands. The membrane was washed thoroughly with water to stop the reaction, and the results were recorded using a digital scanner (Hewlett Packard, Palo Alto, CA).

In the aad-1 protein preparation obtained from P. fluorescens, the main band of the protein that was visualized on Coomassie stained SDS-PAGE gels contained a protein with a molecular weight of approximately 33 kDa. As expected, the corresponding corn-derived aad-1 protein (event DAS-40278-9) was identical in size to the proteins expressed by the microorganisms. As expected, the fractions purified from plants, in addition to the aad-1 protein, contained a small amount of immunologically inactive impurities. Co-purified proteins were likely to remain on the column due to weak interactions with the column matrix or leakage of the monoclonal antibody from the column under severe elution conditions. Other researchers have also reported nonspecific adsorption of peptides and amino acids on immunosorbents based on bromine-activated sepharose 4B (Kennedy and Barnes, 1983; Holroyde et al., 1976; Podlaski and Stern, 2008).

The aad-1 protein obtained from Pseudomonas gave a positive signal of the expected size in a Western blot analysis using a polyclonal antibody. The same signal was also observed in the transgenic corn stalk extract DAS-40278-9. In the aad-1 Western blot analysis, no immunoreactive proteins were found in the control extract of XHH13 and no proteins of alternative sizes (aggregates or degradation products) were observed in the transgenic samples.

Example 5.4. Identification of post-translational glycosylation

Purified by immunoaffinity chromatography, the aad-1 protein obtained from maize (fraction No. 3) was mixed in a 4: 1 ratio with 5x Laemmli buffer. Obtained from aad-1 microorganisms, soybean trypsin inhibitor, bovine serum albumin and horseradish peroxidase were diluted with Milli-Q water to a concentration approximately equal to that obtained from aad-1 plants, and mixed with Laemmli Bio-Rad buffer. Then the proteins were heated at ~ 95 ° C for 5 minutes and centrifuged at 20,000 × g for 2 minutes to obtain a clarified supernatant. The resulting supernatants were directly applied to Bio-RadCriterion gel and electrophoresed using XT MES working buffer (Bio-Rad, Hercules, CA), essentially as described above, except that the electrophoresis was carried out at 170 V for ~ 60 minutes . After electrophoresis, the gel was cut in half, and one half was stained with a blue GelCode dye for total protein according to the manufacturer's protocol. After completion of staining, the gel was scanned on a Molecular Dynamics densitometer, obtaining continuous visual registration of the gel. The other half of the gel was stained using the GelCode glycoprotein staining kit (Pierce Chemical, Rockford, IL) according to the manufacturer's protocol to visualize the glycoproteins. Glycoproteins (with a detection limit of already 0.625 ng per lane) were visualized as purple bands on a light pink background. After the staining of the glycoproteins was completed, the gels were scanned using a Hewlett Packard digital scanner, obtaining continuous visual recording of the gel. After capturing the glycosylation staining image, the gels were stained using GelCode Blue to confirm the presence of non-glycosylated proteins. The results showed that both aad-1 proteins obtained from corn and microorganisms obtained did not have detectable covalently linked carbohydrates. The result was also confirmed by "fingerprints" of peptide masses.

Example 5.5 Fingerprints of peptide masses obtained using mass spectrometry and sequencing of aad-1 obtained from corn and Pseudomonas

Mass spectrometric analysis of aad-1 obtained from Pseudomonas and maize was performed. Protein aad-1, obtained from the stem of transgenic corn (event DAS-40278-9), was digested with trypsin in solution followed by MALDI-TOF-MS and ESI-LC / MS.

Masses of detected peptides were compared with masses calculated based on potential protease cleavage sites in the sequence of aad-1 protein derived from maize. Theoretical cleavage was obtained in silico using the free Protein Analysis Worksheet (PAWS) software from Proteometrics LLC. The protein aad-1 after denaturation is readily cleaved by proteases and will produce numerous peptide peaks.

In the trypsin cleavage product of aad-1 protein obtained from transgenic corn (event DAS-40278-9), the detected peptide fragments covered an almost complete protein sequence, with only one small tryptic fragment at the C-terminus of the protein, from F ²⁴⁸ to R ²⁵³ , missing. and one short (2 amino acids) peptide fragment. This analysis confirmed that the amino acid sequence of the protein obtained from corn coincided with the amino acid sequence of the protein aad-1 obtained from microorganisms. The results of such analyzes show that the amino acid sequence of the aad-1 protein derived from maize was equivalent to the protein expressed by P. fluorescens.

Example 5.5.1. Sequencing tryptic peptide fragments

In addition to fingerprints of the peptide masses, amino acid residues at the N- and C-ends of the aad-1 protein obtained from corn (immunoaffinity purified from corn, in which the DAS-40278-9 event takes place) were sequenced and compared with the sequence of the protein obtained from microorganisms. The protein sequences were obtained using tandem mass spectrometry for the first 11 residues obtained from microorganisms and maize proteins (table 8). The amino acid sequences of both proteins were A ′ HAALSPLSQR ”sequence (SEQ ID NO: 30), indicating that the N-terminal methionine was removed by aminopeptidase (Table 8). The N-terminal sequence of the aad-1 protein was expected to be the sequence M 'AHAALSPLSQ R' ² . (SEQ ID NO: 31). These results indicate that, during or after translation in maize and P. fluorescens, the N-terminal methionine is cleaved by methionine aminopeptidase (MAP). MAPs quickly cleave methionyl residues when the second residue in the protein is small, such as Gly, Ala, Ser, Cys, Thr, Pro, and Val (Walsh, 2006). In addition to the removal of methionine, it was shown that a small portion of the N-terminal peptide of the aad-1 protein was acetylated after cleavage of the N-terminal methionine (Table 8). This result is often encountered in the case of proteins expressed by eukaryotes (plants), since approximately 80-90% of the N-terminal residues are modified (Polevoda and Sherman, 2003). It has also been shown that proteins with serine and alanine at the N-ends are most often acetylated (Polevoda and Sherman, 2002). Two cotranslational processes, the cleavage of the N-terminal methionine residue and N-terminal acetylation, are by far the most common modifications and occur in the case of the vast majority (~ 85%) of eukaryotic proteins (Polevoda and Sherman, 2002). However, examples demonstrating the biological significance associated with N-terminal acetylation are rare (Polevoda and Sherman, 2000).

In addition to N-acetylation, there is also a slight N-terminal shortening that occurs during the purification of the aad-1 protein derived from maize (Table 8). Such “torn ends” resulted in the loss of amino acids A ² , H ³ and A ⁴ (in various forms and quantities) from a protein derived from corn. It is believed that this shortening occurred during the purification of the aad-1 protein, since the Western blot probe of the crude leaf extracts gave one distinct band with the same molecular weight as the aad-1 protein obtained from microorganisms. The extraction buffer for samples subjected to Western blotting contained an excess of a mixture of protease inhibitors, which includes a mixture of protease inhibitors with broad specificity for the inhibition of serine, cysteine, aspartic and metalloproteinases and aminopeptidases.

The C-terminal sequence of aad-1 proteins derived from corn and microorganisms was determined as described above and compared with the expected amino acid sequences (table 9). The results showed that the measured sequences were identical to the expected sequences, and the aad-1 proteins obtained from corn and microorganisms were identical and were not changed at the C-terminus.

Example 6. Field expression, analysis of nutrient composition and agronomic characteristics of the hybrid line of corn, in which the event DAS-40278-9

The aim of this study was to determine the levels of AAD-1 protein found in corn tissue. In addition, we analyzed the composition of corn feed and grain to study the equivalence of the isogenic non-transformed maize line and the transgenic maize line DAS-40278-9 (without spraying, spraying with 2,4-D, spraying with quizalophop and spraying with 2,4-D and quisalophop) . The agronomic characteristics of the isogenic non-transformed maize line were also compared with DAS-40278-9 maize. Field expression, composition, and agronomic studies were conducted at six locations under study in major corn producing regions in the United States and Canada. Such locations represent regions with diverse agronomic practices and varying environmental conditions. Such tests were conducted in Iowa, Illinois (2 locations), Indiana, Nebraska and Ontario in Canada.

The average values at all locations for the control sample, non-sprayed AAD-1 samples, AAD-1 + quisalophop samples, AAD-1 + 2,4-D and AAD-1 + samples were both introduced within the well-known limits for corn. Limited significant differences were observed between unsprayed AAD-1 corn, AAD-1 + corn quizalofop, AAD-1 + 2,4-D or AAD-1 + both and control, but the differences were not considered biologically significant because they were small, and results were within the limits found for commercial corn. The graphs of the results of determining the composition do not show any biologically significant processing-related differences in the composition between the lines of corn: unsprayed AAD-1, AAD-1 + quisalophop, AAD-1 + 2,4-D or AAD-1 + both, and the control a line of corn. In conclusion, the results of the determination of the composition of the corn of uncultivated AAD-1, AAD-1 + quisalophop, AAD-1 + 2,4-D and AAD-1 + both formulations confirm the equivalence of AAD-1 corn (event DAS 40278-9) with the usual lines corn.

Example 6.1. Testing Corn Lines

Hybrid seeds in which the DAS-40278-9 event takes place and control plants in the form of ordinary hybrid seeds with the same genetic background as the line of the test material, but not having the DAS-40278-9 event, are listed in Table 10.

Table 10 Test object Description one non-aad-1 control 2 unsprayed aad-1 3 aad-1 sprinkled with quisalophop four aad-1, sprinkled with 2,4-D 5 aad-1, sprinkled with 2,4-D and quisalophop

The corn plants described above were grown at locations in major corn cultivation regions in the United States and Canada. Six field test sites, Richland, IA; Carlyle, IL; Wyoming, IL; Rockville, IN; York, NE; and Branchton, Ontario, Canada (designated IA, IL1, IL2, IN, NE, and ON) represent regions with diverse agronomic practices and varying environmental conditions for maize.

Test and control corn seeds were sown at a seed rate of approximately 24 seeds per row with a seed spacing of approximately 10 inches (25 cm). In each place, 4 replicates of plots were laid for each treatment, with each plot having rows of 2–25 feet (0.6–7.5 m). The plots were distributed according to a randomized full block (RCB) plan with unique randomization at each location. Each corn plot was limited to 2 rows of a non-transgenic corn hybrid with a similar maturity period. The entire test site was surrounded by at least 12 rows (or 30 feet (9 m)) of a nontransgenic hybrid of corn of similar relative maturity.

Appropriate practical measures were taken to control insects, weeds and diseases in order to obtain a crop corresponding to agronomic requirements. The maximum and minimum monthly temperatures along with rainfall and irrigation were average at each location. These ranges of values are commonly encountered in corn production.

Example 6.2 Herbicide application

Herbicide treatment was carried out using a spray volume of 20 gallons per acre (187 l / ha). Such an introduction was planned in order to reproduce the maximum approved norm in commercial practice. Table 11 lists the herbicides that were used.

Table 11 Herbicide TSN Concentration Weedar 64 026491-0006 39%, ^and 3.76 pounds eq / gallon, 4514 g eq / l Assure ii 106155 10.2%, 0.87 lbs AI ^b / gallon, 104 g AI / L ^a ec = equivalent of acid.
^b ai = active ingredient.

2,4-D (Weedar 64) was applied in the form of 3 applications by scattering from above to test objects 4 and 5 (seasonal only 3 pounds eq / acre). Separate applications were carried out before germination and approximately at stages V4 and V8-V8.5. Planned rates for individual applications were 1.0 lb eq / acre for Weedar 64 or 1120 g eq / ha. Actual application rates were in the range of 1096-1231 g eq / acre.

Quisalofop (Assure II) was applied as a single application by spreading it from above to test objects 3 and 5. The application period was dated approximately to the V6 growth stage. The planned application rate was 0.0825 pounds ai / acre for Assure II or 92 g ai / ha. Actual application rates were in the range of 90.8-103 g ai / ha.

Example 6.3 Collection of agronomic data and results

Agronomic characteristics were recorded for all tested objects in blocks 2, 3, and 4 at each location. Table 12 lists the following measured characteristics.

Table 12 Sign Evaluation time Data Description Early population V1 and V4 The number of plants that appear on the plot Germination V4 Visual assessment of the average thickness of emerged plants on a plot Plant power / damage About 1-2 weeks after application Herbicide damage Time to sweat pistil posts Approximately 50% sweeping of pistil posts The number of accumulated thermal units from the time of sowing to the moment when pistil posts appeared in about 50% of the plants Dusting time Approx. 50% dusting The amount of accumulated thermal units from the time of sowing to the moment when dusting occurs in about 50% of plants Pollen vitality About 50% Assessing the color and shape of pollen over time Plant height About R6 Height to panicle tip Cob Height About R6 Height to the base of the primary cob Stem lodging About R6 Visual assessment of the percentage of plants on a plot with stems broken below the primary cob Root lodging About R6 Visual assessment of the percentage of plants in the plot with lodging approximately 30 ° or more in the first ~ 1/2 meter above the soil surface Final population About R6 The number of plants remaining in the plot Days to ripen About R6 The number of accumulated thermal units from the time of sowing to the moment when approximately 50% of the plants reach physiological maturity Preservation of green coloring About R6 General condition of the plant Incidence About R6 Visual assessment of the frequency of leaf disease Insect damage About R6 Visual assessment of insect damage Note: Thermal unit = ((maximum temperature + minimum temperature) / 2) -50 ° F.

The agronomic data collected for the following objects were analyzed: control, aad-1 unsprayed, aad-1 + 2,4-D, aad-1 + quisalofop, and aad-1 + both. In the case of analysis in different regions, no statistically significant differences were observed in the values for early populations (V1 and V4), power, end populations, culture damage, time of sweeping of pistillar columns, dusting time, stem lodging, root lodging, incidence, insect damage, number days before ripening, plant height and pollen viability (shape and color), judging by the total analysis in different regions (table 13). In the case of the study of the ability to maintain green color and heights to the cob, significant differences were observed when paired t-criteria were used between the control and the aad-1 + quisalophop object, but they were not accompanied by significant general processing effects or p-values corrected for the average share of false deviations from hypothesis (FDR) (table 13).

Table 13 The total analysis of the results of the study of agronomic characteristics in different locations for corn aad-1 DAS-40278-9 (sprinkled and unsprayed) and control Analit The overall treatment effect (Pr> F) ^a The control Unsprayed (P-value, ^b adjusted P) ^c Sprayed with quisalophop, p (P-value, adjusted P) Sprinkled 2,4-D (P-value, adjusted P) Sprayed with two drugs (P-value, adjusted P) Early V1 population (number of plants) (0,351) 42.8 41.3
(0.303, 0.819) 41.7
(0.443, 0.819) 41.9
(0.556, 0.819) 44.1
(0.393, 0.819) Early V4 population (number of plants) (0.768) 43.1 43.3
(0.883, 0.984) 43.7
(0.687, 0.863) 44.3
(0.423, 0.819) 44.8
(0.263, 0.819) Germination ^d (0,308) 7.69 7.39
(0.197, 0.819) 7.36
(0.161, 0.819) 7.58
(0.633, 0.819) 7.78
(0.729, 0.889) Final population (number of plants) (0,873) 40.1 39.6
(0.747, 0.889) 39.7
(0.802, 0.924) 39.9
(0.943, 1.00) 41.1
(0.521, 0.819) Culture damage - 1st application ^e NA ^l 0 0 0 0 0 Culture damage - 2nd application ^e (0.431) 0 0
(1.00, 1.00) 0
(1.00, 1.00) 0
(1.00, 1.00) 0.28
(0.130, 0.819) Culture damage - 3rd application ^e NA 0 0 0 0 0 Culture damage - 4th application ^e NA 0 0 0 0 0 Time to sweat pistil posts (thermal units) ^f (0.294) 1291 1291
(0.996, 1.00) 1293
(0.781, 0.917) 1304
(0,088, 0,819) 1300
(0.224, 0.819) Time to dusting (thermal units) ^f (0,331) 1336 1331
(0.564, 0.819) 1342
(0.480, 0.819) 1347
(0.245, 0.819) 1347
(0.245, 0.819) Pollen form 0 minutes (%) ^g (0.872) 10.9 10.9
(0.931, 1.00) 11.3
(0.546, 0.819) 11,4
(0.439, 0.819) 11.3
(0.605, 0.819) Pollen form 30 minutes (%) (0.486) 49.2 50.8
(0.618, 0.819) 46,4
(0.409, 0.819) 48.1
(0.739, 0.889) 51.9
(0.409, 0.819) Pollen shape 60 minutes (%) (0.724) 74,4 74.7
(0.809, 0.924) 73.6
(0.470, 0.819) 73.9
(0.629, 0.819) 75.0
(0.629, 0.819) Pollen shape 120 minutes (%) (0.816) 82.6 82.6
(1.00, 1.00) 82.6
(1.00, 1.00) 82.6
(1.00, 1.00)
82.5
(0.337, 0.819) Pollen color 30 minutes (%) (0,524) 51.9 52,5
(0.850, 0.960) 48.9
(0.306, 0.819) 50.3
(0.573, 0.819) 53.6
(0.573, 0.819) Pollen color 60 minutes (%) (0.332) 75.3 75.9
(0.612, 0.819) 74,2
(0.315, 0.819) 74,2
(0.315, 0.819) 75.9
(0.612, 0.819) Pollen color 120 minutes (%) NA 84.0 84.0 84.0 84.0 84.0 Stem lodging (%) (0.261) 5.11 5.22
(0.356, 0.819) 5.00
(0.356, 0.819)
5.00
(0.356, 0.819) 5.00
(0.356, 0.819) Root lodging (%) (0.431) 0.44 0.17
(0.457, 0.819) 0.72
(0.457, 0.819)
0.17
(0.457, 0.819) 0.11
(0.373, 0.819) Preserving green coloring ⁱ (0.260) 4.67 4.28
(0.250, 0.819) 3.92
(0,034 ^m , 0,819) 4.17
(0.144, 0.819) 4.11
(0.106, 0.819) Incidence ^j (0.741) 6.42 6.22
(0.383, 0.819) 6.17
(0.265, 0.819) 6.17
(0.265, 0.819)
6.17
(0.265, 0.819) Insect damage ^k (0.627) 7.67 7.78
(0,500, 0,819)
7.78
(0,500, 0,819) 7.72
(0.736, 0.889) 7.56
(0,500, 0,819) Days to maturity (thermal units) ^f (0.487) 2411 2413
(0.558, 0.819)
2415
(0.302, 0.819)
2416
(0.185, 0.819)
2417
(0.104, 0.819) Plant height (cm) (0.676) 294 290
(0.206, 0.819) 290
(0.209, 0.819) 291
(0.350, 0.819) 291
(0.286, 0.819) Height to the cob (cm) (0,089) 124 120
(0,089, 0,819) 118
(0.018 ^m , 0.786) 121
(0.214, 0.819) 118
(0.016 ^m , 0.786) ^a Overall treatment effect evaluated using the F-test.
^b Comparison of sprayed and non-sprayed treatments with control using the t-test.
^c P-values corrected using the method for estimating the average proportion of false hypothesis deviations (FDR).
^d Visual rating on a scale of 1–9; 9 = tall plants with large strong leaves.
^e Scale 0-100%; 0 = no damage and 100 = dead plant.
^{f The} number of thermal units that have been accumulated since sowing.
^g Scale 0-100%; % pollen grains with destroyed walls.
^h Scale 0-100%; % pollen grains with intense yellow color.
ⁱ Visual rating on a scale of 1–9, where 1 means no visible green tissue.
^j Visual rating on a scale of 1-9, where 1 means poor resistance to disease.
^k Visual rating on a scale of 1–9, where 1 means poor insect resistance.
^l NA = no statistical analysis was carried out since there was no variability between repetitions or treatments.
^m Statistical difference is shown by a P-value <0.05.

Example 6.4 Sample Collection

Samples for expression and analysis of the composition were collected as indicated in table 14.

Example 6.5 Determination of aad-1 protein in maize samples

Corn samples were analyzed in relation to the amount of aad-1 protein. The aad-1 soluble extractable protein was quantified using an enzyme-linked immunosorbent assay (ELISA) kit purchased from Beacon Analytical System, Inc. (Portland, ME).

Corn tissue samples were isolated from the test plants and prepared for expression analysis using coarse grinding, lyophilization and fine grinding (if necessary) using a Geno / Grinder grinder (Certiprep, Metuchen, New Jersey). For pollen, no additional preparation was required. Protein aad-1 was extracted from corn tissue with a solution of phosphate-buffered saline containing tween-20 detergent (PBST) and containing 0.5% bovine serum albumin (BSA). In the case of pollen, the protein was extracted using 0.5% PBST / BSA buffer containing 1 mg / ml sodium ascorbate and a 2% mixture of protease inhibitors. Plant tissue and pollen extracts were centrifuged; an aqueous supernatant was collected, if necessary, diluted with an appropriate buffer and analyzed using a sandwich ELISA kit aad-1. The kit was used in the following steps. An aliquot of the diluted sample and biotinylated monoclonal anti-aad-1 antibody were incubated in the wells of a microtiter plate coated with immobilized monoclonal anti-aad-1 antibody. Such antibodies bind to the aad-1 protein in the wells and form a “sandwich” with the aad-1 protein, which is bound between the soluble and immobilized antibody. Unbound samples and conjugate were then removed from the plate by washing with PBST. Excess streptavidin-enzyme conjugate (alkaline phosphatase) was added to the wells for incubation. At the end of the incubation period, unbound reagents were removed from the plate by washing. Subsequent addition of the enzyme substrate resulted in the formation of a colored product. Since aad-1 was bound in the antibody sandwich, the level of color development was associated with the concentration of aad-1 in the sample (i.e., lower residue concentrations resulted in less color development). Absorption at 405 nm was measured using a Molecular Devices V-max or Spectra Max 190 plate reader. A calibration curve was obtained and the aad-1 concentration in unknown samples was calculated from the polynomial regression equation using Soft-MAX Pro ^TM software that was compatible with a tablet reader. Samples were analyzed using wells in duplicate, with the average concentration reported in duplicate wells.

The total data on the concentration of aad-1 protein (averaged for different regions) in various forms of corn are shown in table 15. The average concentration of aad-1 protein was in the range from 2.87 ng / mg dry weight in the root at the stage R1 to 127 ng / mg in pollen. The results of the determination of expression for non-sprayed and sprayed plots were similar. The aad-1 protein was not detected in any of the control samples, with the exception of one control root sample from Indiana.

Table 15 Summary data on average levels of aad-1 protein concentration measured in maize tissue: unaddressed aad-1, aad-1 + quisalophop, aad-1 + 2,4-D and aad-1 + quisalophop and 2,4-D Corn cloth Treatment AAD-1 ng / mg dry tissue mass Average Standard
deviation Range V2-V4 sheet AAD-1 non-sprayable 13,4 8.00 1.98-29.9 AAD-1 + Quisalophop 13.3 6.89 4.75-24.5 AAD-1 + 2,4-D 14.2 7.16 4.98-26.7 AAD-1 + Quisalophop and 2,4-D 12.3 7.09 4.07-22.5 V9 sheet AAD-1 non-sprayable 5.96 2,50 2.67-10.9 AAD-1 + Quisalophop 5.38 1.84 2,52-9,15 AAD-1 + 2,4-D 6.37 2.41 3.03-10.9 AAD-1 + Quisalophop and 2,4-D 6.52 2,38 3.11-11.1 R1 sheet AAD-1 non-sprayable 5.57 1.66 3.47-9.34 AAD-1 + Quisalophop 5.70 1,63 2.70-7.78 AAD-1 + 2,4-D 5.99 1.90 2.40-9.42 AAD-1 + Quisalophop and 2,4-D 6.06 2.27 1.55-10.2 Pollen AAD-1 non-sprayable 127 36,2 56.3-210 AAD-1 + Quisalophop 108 29.9 52,2-146 AAD-1 + 2,4-D 113 30,2 37.5-137 AAD-1 + Quisalophop and 2,4-D 112 32.6 45.4-162 R1 root AAD-1 non-sprayable 2.92 1.87 0.42-6.10 AAD-1 + Quisalophop 3.09 1.80 0.56-6.06 AAD-1 + 2,4-D 3.92 2.03 0.91-7.62 AAD-1 + Quisalophop and 2,4-D 2.87 1.23 1.09-5.56 R4 forage AAD-1 non-sprayable 6.87 2.79 2,37-12,1 AAD-1 + Quisalophop 7.16 2.84 3.05-11.6 AAD-1 + 2,4-D 7.32 2.46 2.36-10.6 AAD-1 + Quisalophop and 2,4-D 6.84 2,31 2.25-10.3 Whole plant AAD-1 non-sprayable 4,53 2,55 0.78-8.88 AAD-1 + Quisalophop 4.61 2.22 0.75-8.77 AAD-1 + 2,4-D 5.16 2,53 0.83-10.2 AAD-1 + Quisalophop and 2,4-D 4,55 1.77 1.30-8.21 Corn AAD-1 non-sprayable 5.00 1,53 2.66-8.36 AAD-1 + Quisalophop 4.63 1.51 1.07-6.84 AAD-1 + 2,4-D 4.98 1.78 2.94-9.10 AAD-1 + Quisalophop 2,4-D 4.61 1,62 1.81-7.49 ^a ND = value less than the sensitivity limit (LOD) of the method.
^b Values in parentheses between the method LOD and the quantification limit (LOQ).

Example 6.6 Composition analysis

Maize fodder and grain samples were analyzed for nutrient content using various tests. Analyzes carried out in the case of fodder included analysis of ash, total fat, moisture, proteins, carbohydrates, coarse fibers, acid detergent fiber, neutral detergent fiber, calcium and phosphorus. Analyzes carried out in the case of grain included analyzes of the main components of food (ash, total fat, moisture, proteins, carbohydrates, coarse fibers, acid detergent fiber), neutral detergent fiber (NDF), minerals, amino acids, fatty acids, vitamins, secondary metabolites and anti-nutrients. Nutrient analysis results for corn fodder and grain were compared with the values reported in the literature (see; Watson, 1982 (4); Watson, 1984 (5); ILSI Crop Composition Database, 2006 (6); OECD Consensus Document on Compositional Considerations for maize, 2002 (7); and Codex Alimentarius Commission 2001 (8)).

Example 6.6.1. Analysis of nutrients, fibers and minerals in fodder

We analyzed proteins, fat, ash, moisture, carbohydrates, ADF, NDF, calcium and phosphorus in samples of corn fodder obtained from the following corn plants: control, non-sprayed aad-1, aad-1 + quizalofop, aad-1 +2.4 -D and aad-1 + both drugs. The total results for all regions are shown in Table 16. In the case of analysis for all regions and analysis in individual regions, all average values for nutrients, fibers and minerals were within the limits known from the literature. No statistical differences were observed in the analysis for different regions between the control and transgenic objects in terms of moisture content, ADF, NDF, calcium and phosphate. Regarding protein and ash content, significant differences were observed when using paired t-criteria for non-sprayed AAD-1 (protein), aad-1 + quisalophop (protein) and aad-1 + both drugs (ash), but no significant general effects were observed processing or p-values adjusted according to FDR. With respect to the study of fat, significant differences were observed when using the paired t-test and the adjusted p-value in the case of aad-1 + quisalophop compared to the control, but no significant overall treatment effect was observed. For carbohydrates, a statistically significant overall treatment effect, paired t-test and FDR-adjusted p-value were observed when comparing aad-1 + quisalophop and control. Also, for carbohydrates, a significant difference was observed when using the paired t-test in the case of non-sprayed aad-1 plants, but without a significant FDR-corrected p-value. Such differences are not biologically significant, since the results obtained in different regions for such analytes were in the ranges reported in the literature for corn, and differences from the control were small (<23%).

Table 16 Summary analysis of nutrient components, fibers and minerals in corn fodder from all locations Nutrient components (% of dry weight) Literary Values ^a The overall effect of processing
(Pr> F) ^b The control Unsprayed (P-value, ^c adjusted P) ^d Sprayed with quisalophop (P-value, adjusted P) Sprinkled 2,4-D (P-value, adjusted P) Sprayed with two drugs (P-value, adjusted P) Protein 3.14-15.9 (0,054) 7.65 6.51
(0.016 ^e , 0.066) 6.41
(0.010 ^e ,
0.051) 7.17
(0.285, 0.450) 7.13
(0.245, 0.402) Fat 0.296-6.7 (0,068) 2.29 2.08
(0.202, 0.357) 1.78
(0.005 ^e ,
0.028 ^e ) 2.10
(0.233, 0.391) 2.01
(0,093, 0,213) Ash 1.3-10.5 (0,072) 3.90 3.84
(0.742, 0.859) 4.03
(0.525,
0.708) 3.99
(0.673, 0.799) 4.40
(0.019 ^e , 0.069) Humidity 53.3-87.5 (0.819) 69.5 69.2
(0.651, 0.782) 69.5
(0.988,
0.988) 69.8
(0.699, 0.820) 70.0
(0.501, 0.687) Carbohydrates 66.9-94.5 (0,026 ^e ) 86.1 87.6
(0.015 ^e , 0.061) 87.8
(0.006 ^e ,
0.034 ^e ) 86.8
(0.262, 0.424) 86.5
(0.538, 0.708) Fibers (% of dry weight) Acid Detergent Fiber (ADF) 16.1-47.4 (0.968) 26.5 26.6
(0.925, 0.970) 26.8
(0.833,
0.925) 26.0
(0.677, 0.800) 26.8
(0.851, 0.937) Neutral Detergent Fiber (NDF) 20.3-63.7 (0.345) 41.6 43.6
(0.169, 0.322) 43.3
(0.242,
0.402) 41.3
(0.809, 0.911) 41.6
(0.978, 0.985) Minerals (% of dry weight) Calcium 0.071-0.6 (0,321) 0.212 0,203
(0.532, 0.708) 0.210
(0.930,
0.970) 0.215
(0.815, 0.911) 0.231
(0.150, 0.296) Phosphorus 0.094-0.55 (0,163) 0.197 0.189
(0.198, 0.354) 0.202
(0.427,
0.615) 0,203
(0.288, 0.450) 0,200
(0.608, 0.762) ^a combined range.
^b Overall treatment effect evaluated using the F-test.
^c Comparison of transgenic treatments with control using t-test.
^d P-values corrected using the method for estimating the average fraction of false hypothesis deviations (FDR).
^e Statistical difference is indicated by a P-value <0.05.

Example 6.6.2. Analysis of nutrients and fiber in grain

The total results of the analysis of nutrient components (protein, fat, ash, moisture, cholesterol and carbohydrates) and fibers (ADF, NDF and total dietary fiber) in corn grain in all locations are shown in table 17. All results for nutrient components and fibers were in ranges known from the literature, and no significant differences were observed in the analysis at different locations between the control and transgenic objects with respect to fat, ash, NDF and total dietary fiber. With respect to moisture content, a significant overall treatment effect was observed, but it was not accompanied by significant paired t-criteria or FDR-corrected p-values. For ADF, a significant paired t-test was observed for both aad-1 + drugs, but no significant overall treatment effect or FDR-corrected p-value was observed. For protein and carbohydrates, significant differences were obtained using paired t-criteria, adjusted p-values and overall treatment effects in the case of the unadulterated aad-1, aad-1 + quisalofop and aad-1 + both. Since such differences were small (<12%) and all values were in the ranges known from the literature, such differences were not considered biologically important.

Table 17 Summary analysis of nutrient components and fibers in corn grain from all locations Nutrient components (% of dry weight) Literary Values ^a The overall effect of processing
(Pr> F) ^b The control Unsprayed (P-value, ^c adjusted P) ^d Sprayed with quisalophop (P-value, adjusted P) Sprinkled 2,4-D (P-value, adjusted P) Sprayed with two drugs (P-value, adjusted P) Protein 6-17.3 (0.003 ^e ) 9.97 10.9
(0.002 ^e , 0.016 ^e ) 11.1
(0.0004 ^e , 0.013 ^e ) 10.5
(0,061, 0,161) 10.9
(0.002 ^e , 0.015 ^e ) Fat 1,2-18,8 (0.369) 4.26 4.19
(0.238, 0.397) 4.16
(0,095, 0,215) 4.26
(0.955, 0.977) 4.22
(0.427, 0.615) Ash 0.62-6.28 (0,553) 1.45 1.55
(0.178, 0.330) 1,52
(0.364, 0.557) 1.45
(0.982, 0.985) 1.51
(0.397, 0.587) Humidity 6.1-40.5 (0,038 ^e ) 25.1 25.5
(0.406, 0.594) 24.4
(0.056, 0.152) 24.5
(0,117, 0,254) 24.5
(0.114, 0.250) Cholesterol NR ^f NA ^g <LOQ <LOQ <LOQ <LOQ <LOQ Carbohydrates 63.3-89.8 (0.005 ^e ) 84.3 83.3
(0.002 ^e , 0.015 ^e ) 83,2
(0.001 ^e ,
0.013 ^e ) 83.8
(0,074, 0,185) 83,4
(0.003 ^e , 0.019 ^e ) Fibers (% of dry weight) Acid Detergent Fiber (ADF) 1.82-11.3 (0.247) 4.23 3.94
(0.130, 0.269) 3.99
(0.197,
0.354) 3.89
(0,078, 0,193) 3.82
(0.035 ^e , 0.106) Neutral Detergent Fiber (NDF) 5.59-22.6 (0.442) 10.6 10.3
(0.455, 0.638) 9.89
(0.120, 0.254) 9.90
(0,121, 0,254) 10.3
(0.552, 0.708) Common Dietary Fiber 8.3-35.3 (0,579) 13,4 12.8
(0.164, 0.313) 12.9
(0.195, 0.353) 13.1
(0.487, 0.679) 12.9
(0.215, 0.370) ^a combined range.
^b Overall treatment effect evaluated using the F-test.
^c Comparison of transgenic treatments with control using t-test.
^d P-values corrected using the method for estimating the average fraction of false hypothesis deviations (FDR).
^e Statistical difference is indicated by a P-value <0.05.
^f NR = not reported.
^g NA = no statistical analysis was performed as most of the data was <LOQ.

Example 6.6.3. Analysis of minerals in grain

Samples of corn grains were analyzed for minerals of calcium, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, sodium and zinc. The total results obtained at all locations are shown in Table 18. All results were in the ranges known from the literature. In the case of analysis at different locations, no significant differences were observed with respect to calcium, copper, iron and potassium. The average results for chromium, iodine, selenium and sodium were below the limit of quantitation in this way. With respect to magnesium and phosphorus, significant paired t-criteria were observed for the non-sprayed aad-1 plants and aad-1 + quisalophop plants, but they were not accompanied by significant overall treatment effects or FDR-corrected p-values. With respect to manganese and molybdenum, a significant paired t-test was observed for unadultivated aad-1 plants, but no FDR-corrected p-value and overall treatment effect were found. In the case of aad-1 +, both drugs observed a significant paired t-test for zinc, but no significant FDR-corrected p-value or overall treatment effect. In addition, such differences from the control were small (<13%), and all values were in the ranges known from the literature when they were available.

Table 18 Total analysis of minerals in corn grain from all locations Minerals (mg / 100 g dry weight) Literary Values ^a The overall effect of processing
(Pr> F) ^b The control Unsprayed (P-value, ^c adjusted P) ^d Sprayed with quisalophop (P-value, adjusted P) Sprinkled 2,4-D (P-value, adjusted P) Sprayed with two drugs (P-value, adjusted P) Calcium 1.27-100 (0.493) 4.05 4.21
(0.146, 0.289) 4.12
(0.505, 0.687) 4.04
(0.944, 0.977) 4.06
(0.898, 0.957) Chromium 0.006-0.016 NA ^e <LOQ <LOQ <LOQ <LOQ <LOQ Copper 0.073-1.85 (0.963) 0.144 0.151
(0.655, 0.782) 0.146
(0.890,
0.957) 0.141
(0.817, 0.911) 0.149
(0.749, 0.863) Iodine 7.3-81 NA <LOQ <LOQ <LOQ <LOQ <LOQ Iron 0,1-10 (0.333) 2.49 2.60
(0,086, 0,206) 2,56
(0.310,
0.482) 2,51
(0.801, 0.911) 2.59
(0.145, 0.289) Magnesium 59.4-1000 (0,072) 122 129
(0.010 ^f , 0.051) 128
(0.017 ^f ,
0,066) 126
(0.145, 0.289) 127
(0,070, 0,177) Manganese 0,07-5,4 (0,099) 0.525 0.551
(0,025 ^f , 0,082) 0.524
(0.884, 0.957) 0.526
(0.942, 0.977) 0.532
(0.505, 0.687) Molybdenum Nr (0,143) 261 229
(0,020 ^f , 0,072) 236
(0,067, 0,173) 244
(0.206, 0.362) 234
(0,046, 0,132) Phosphorus 147-750 (0.102) 289 303
(0.012 ^f , 0.057) 300
(0,035 ^f ,
0.106) 299
(0,055, 0,150) 298
(0,085, 0,206) Potassium 181-720 (0.453) 362 368
(0.330, 0.510) 359
(0.655,
0.782) 364
(0.722, 0.839) 357
(0.454, 0.638) Selenium 0.001-0.1 NA <LOQ <LOQ <LOQ <LOQ <LOQ Sodium 0-150 NA <LOQ <LOQ <LOQ <LOQ <LOQ Zinc 0.65-3.72 (0,166) 2.26 2,32
(0.183, 0.336) 2,34
(0.108,
0.238) 2.29
(0.627, 0.768) 2,37
(0,027 ^f , 0,085) ^a combined range.
^b Overall treatment effect evaluated using the F-test.
^c Comparison of transgenic treatments with control using t-test.
^d P-values corrected using the method for estimating the average fraction of false hypothesis deviations (FDR).
^e NA = no statistical analysis was performed as most of the data was <LOQ.
^f Statistical difference is shown by a P-value <0.05.

Example 6.6.4. Analysis of amino acids in grain

Corn samples were analyzed with respect to the amino acid content in the control, in unaddressed aad-1 corn plants, in aad-1 + quisalophop plants, aad-1 + 2,4-D and aad-1 + both preparations, and the total results for all regions and the fields at individual locations are shown in Table 19. The levels of all amino acids were in the ranges reported in the literature and there were no significant differences in analysis at different locations for arginine, lysine, and tyrosine. Significant differences were observed for several amino acids in the analysis at different locations. In such cases, the amino acid content in the control was lower than in aad-1 transgenic lines, which may be associated with a lower total protein content in the control grain compared to aad-1 lines. In the case of the non-sprinkled test object, aad-1 found significant common processing effects along with significant paired t-criteria and obtained FDR-corrected p-values for all amino acids, except arginine, glycine, lysine, tryptophan and tyrosine. In the case of aad-1 + quisalofop, they found significant common processing effects along with significant paired t-criteria and obtained FDR-corrected p-values for all amino acids except arginine, cysteine, glycine, lysine, tryptophan and tyrosine. In the case of the aad-1 + 2,4-D object, significant common processing effects were found along with significant paired t-criteria (with significant FDR-adjusted p-values) for all amino acids, except arginine, aspartic acid, glycine, histidine, lysine tyrosine and valine. In the case of the aad-1 + object, both drugs found significant common treatment effects along with significant paired t-criteria and FDR-corrected p-values for all amino acids, except arginine, glycine, lysine, serine, tryptophan and tyrosine. Although there were many differences observed for amino acids, the differences were small (<15%), but were not observed between different locations, and all averages were in the ranges reported in the literature.

Table 19 The total analysis of amino acids in corn grain from all locations Amino acids (% of dry weight) Literary Values ^a The overall effect of processing
(Pr> F) ^b The control Unsprayed (P-value, ^c adjusted P) ^d Sprayed with quisalophop (P-value, adjusted P) Sprinkled 2,4-D (P-value, adjusted P) Sprayed with two drugs (P-value, adjusted P) Alanine 0.44-1.39 (0.002 ^e ) 0.806 0.901
(0.0005 ^e , 0.013 ^e ) 0,900
(0.0005 ^e , 0.013 ^e ) 0.863
(0,021 ^e , 0,074) 0.894
(0.001 ^e , 0.013 ^e ) Arginine 0.12-0.64 (0.371) 0.486 0.499
(0.286, 0.450) 0.505
(0.139,
0.283) 0.487
(0.929, 0.970) 0.484
(0.897, 0.957) Aspartic acid 0.34-1.21 (0.010 ^e ) 0.712 0.768
(0.002 ^e , 0.015 ^e ) 0.764
(0.003 ^e ,
0.021 ^e ) 0.743
(0,060, 0,160) 0.762
(0.004 ^e , 0.027 ^e ) Cysteine 0.08-0.51 (0,033 ^e ) 0.213 0.225
(0.009 ^e , 0.050 ^e ) 0.223
(0,020 ^e ,
0,072) 0.223
(0.018 ^e , 0.067) 0.226
(0.005 ^e , 0.028 ^e ) Glutamic acid 0.97-3.54 (0.001 ^e ) 1.97 2.22
(0.0003 ^e , 0.013 ^e ) 2.21
(0.0004 ^e , 0.013 ^e ) 2.12
(0.017 ^e , 0.067) 2.20
(0.001 ^e , 0.013 ^e ) Glycine 0.18-0.54 (0,052) 0.383 0.397
(0.018 ^e , 0.067) 0.398
(0.013 ^e ,
0,059) 0.390
(0.217, 0.371) 0.397
(0.016 ^e , 0.066) Histidine 0.14-0.43 (0.005 ^e ) 0.283 0,303
(0.001 ^e , 0.013 ^e ) 0.302
(0.002 ^e ,
0.014 ^e ) 0.295
(0,036, 0,109) 0.302
(0.002 ^e , 0.014 ^e ) Isoleucine 0.18-0.71 (0.003 ^e ) 0.386 0.427
(0.001 ^e , 0.014 ^e ) 0.427
(0.001 ^e ,
0.014 ^e ) 0.410
(0,044 ^e , 0,127) 0.431
(0.001 ^e , 0.013 ^e ) Leucine 0.64-2.49 (0.001 ^e ) 1.35 1,54
(0.0003 ^e , 0.013 ^e ) 1,54
(0.0003 ^e , 0.013 ^e ) 1.47
(0.013 ^e , 0.059) 1,53
(0.001 ^e , 0.013 ^e ) Lysine 0.05-0.56 (0.211) 0.310 0.315
(0.210, 0.367) 0.316
(0,128,
0.265) 0,309
(0.879, 0.956) 0.316
(0.102, 0.226) Methionine 0.10-0.47 (0.003 ^e ) 0.195 0.209
(0.001 ^e , 0.013 ^e ) 0.209
(0.001 ^e ,
0.013 ^e ) 0.205
(0.014 ^e , 0.061) 0.208
(0.001 ^e , 0.014 ^e ) Phenylalanine 0.24-0.93 (0.002 ^e ) 0.551 0.617
(0.001 ^e , 0.013 ^e ) 0.619
(0.001 ^e ,
0.013 ^e ) 0.592
(0,023 ^e , 0,077) 0.615
(0.001 ^e , 0.013 ^e ) Proline 0.46-1.63 (0.002 ^e ) 0.910 1.01
(0.0004 ^e , 0.013 ^e ) 1.01
(0.001 ^e ,
0.013 ^e ) 0.975
(0.012 ^e , 0.059) 0,997
(0.001 ^e , 0.014 ^e ) Serine 0.24-0.91 (0.009 ^e ) 0.498 0.550
(0.002 ^e , 0.014 ^e ) 0.550
(0.001 ^e ,
0.014 ^e ) 0.529
(0,042 ^e , 0,122) 0.536
(0.015 ^e , 0.061) Threonine 0.22-0.67 (0.005 ^e ) 0.364 0.394
(0.001 ^e , 0.014 ^e ) 0.394
(0.001 ^e ,
0.013 ^e ) 0.384
(0,023 ^e , 0,077) 0.390
(0.003 ^e , 0.020 ^e ) Tryptophan 0.03-0.22 (0,088) 0,052 0,055
(0,067, 0,173) 0.056
(0,025 ^e ,
0,082) 0.056
(0.014 ^e , 0.060) 0.056
(0,029 ^e , 0,092) Tyrosine 0.10-0.79 (0.390) 0.336 0.355
(0.535, 0.708) 0.375
(0.214,
0.370) 0.339
(0.907, 0.964) 0.314
(0,500, 0,687) Valine 0.21-0.86 (0.005 ^e ) 0.495 0.537
(0.002 ^e , 0.014 ^e ) 0.538
(0.002 ^e ,
0.014 ^e ) 0.519
(0,054, 0,148) 0.538
(0.001 ^e , 0.014 ^e ) ^a combined range.
^b Overall treatment effect evaluated using the F-test.
^c Comparison of transgenic treatments with control using t-criteria.
^d P-values corrected using the method for estimating the average fraction of false hypothesis deviations (FDR).
^e Statistical difference is indicated by a P-value <0.05.

Example 6.6.5. Grain Fatty Acid Analysis

The analysis of samples of corn grain in relation to fatty acids. The total results obtained at different locations are shown in Table 20. All results for control maize samples, unsprayed aad-1 corn plants, aad-1 + quisalofop, aad-1 + 2,4-D and aad-1 + both analyzed in relation to fatty acids were in the ranges published in the literature. Results for caprylic (8: 0), capric (10: 0), lauric (12: 0), myristic (14: 0), myristic (14: 1), pentadecanoic (15: 0), pentadecene (15: 1) , heptadecanoic (17: 0), heptadecenoic (17: 1), gamma-linolenic (18: 3), eicosadiene (20: 2), eicosatrienoic (20: 3) and arachidonic (20: 4) acids were below the limit of quantification (LOQ) method. In the analysis at different locations, no differences were found for 16: 0 palmitic, 16: 1 palmitic, 18: 0 stearic, 18: 2 linoleic, 18: 3 linolenic and 20: 0 arachidonic acids. With respect to 18: 1 oleic and 20: 1 eicosenoic acids, significant paired t-criteria were observed in the case of unsprayed corn aad-1 (18: 1) and corn aad-1 + 2,4-D (18: 1 and 20: 1) , but they were not accompanied by significant general processing effects or FDR-adjusted p-values. With a 22: 0 ratio of behenic acid, a significant overall treatment effect and significant paired t-criteria were found for aad-1 + 2,4-D and aad-1 + both, but there were no significant FDR-corrected p-values.

Table 20 Total analysis of fatty acids in corn grain from all locations Fatty acids (% of total fatty acids) ^a Literary Values ^b The overall effect of processing
(Pr> F) ^c The control Unsprayed (P-value, ^d adjusted P) ^c Sprayed with quisalophop (P-value, adjusted P) Sprinkled 2,4-D (P-value, adjusted P) Sprayed with two drugs (P-value, adjusted P) 8: 0 caprylic 0.13–0.34 NA ^f <LOQ <LOQ <LOQ <LOQ <LOQ 10: 0 capric Nd NA <LOQ <LOQ <LOQ <LOQ <LOQ 12: 0 lauric ND –0.687 NA <LOQ <LOQ <LOQ <LOQ <LOQ 14: 0 myristic ND-0.3 NA <LOQ <LOQ <LOQ <LOQ <LOQ 14: 1 myristol Nr NA <LOQ <LOQ <LOQ <LOQ <LOQ 15: 0 pentadecane Nr NA <LOQ <LOQ <LOQ <LOQ <LOQ 15: 1 pentadecene Nr NA <LOQ <LOQ <LOQ <LOQ <LOQ 16: 0 palmitic 7–20.7 (0,559) 9.83 9.88
(0.618, 0.763) 9.95
(0.280,
0.445) 9.78
(0.617, 0.763) 9.90
(0.544, 0.708) 16: 1 palmitoleic ND – 1.0 (0,552) 0.056 0,044
(0.804, 0.911) 0,047
(0.551,
0.708) 0,041
(0.555, 0.708) 0,079
(0.392, 0.582) 17: 0 heptadecano ND – 0.11 NA <LOQ <LOQ <LOQ <LOQ <LOQ 17: 1 heptadecene ND – 0.1 NA <LOQ <LOQ <LOQ <LOQ <LOQ 18: 0 stearic Nd-3.4 (0.561) 2.04 1.98
(0.119, 0.254) 2.01
(0.437,
0.626) 2.00
(0.259, 0.421) 2.02
(0.598, 0.756) 18: 1 oleic 17.4-46 (0,076) 31.3 30,4
(0.013 ^g , 0.059) 30.8
(0.178,
0.329) 30,4
(0.015 ^g , 0.061) 30.7
(0,092, 0,213) 18: 2 linoleic 34.0-70 (0.474) 47.5 48.3
(0.189, 0.345) 48,4
(0.144,
0.289) 48.0
(0.453, 0.638) 48.5
(0.119, 0.254) 18: 3 gamma-linolenic Nr NA <LOQ <LOQ <LOQ <LOQ <LOQ 18: 3 linolenic Nd-2.25 (0.479) 1,04 1.05
(0.537, 0.708) 1.06
(0.202,
0.357) 1,04
(0.842, 0.932) 1.06
(0.266, 0.428) 20: 0 arachidonic 0.1-2 (0,379) 0.400 0.386
(0,061, 0,161) 0.393
(0.341,
0.525) 0.390
(0.153, 0.297) 0.390
(0.175, 0.328) 20: 1 eicosene 0.17–1.92 (0.107) 0.232 0.226
(0,089, 0,210) 0.230
(0.497,
0.687) 0.223
(0.013 ^g , 0.059) 0.227
(0,121, 0,254) 20: 2 eicosadiene ND – 0.53 NA <LOQ <LOQ <LOQ <LOQ <LOQ 20: 3 eicosatrienoid 0.275 NA <LOQ <LOQ <LOQ <LOQ <LOQ 20: 4 arachidonic 0.465 NA <LOQ <LOQ <LOQ <LOQ <LOQ 22: 0 beige ND – 0.5 (0,044 ^g ) 0.136 0,088
(0,093, 0,213) 0,076
(0.887,
0.957) 0,086
(0.011 ^g , 0.054) 0.108
(0,023 ^g , 0,077) ^a Results converted from units% of dry weight to% of fatty acids.
^b Combined range.
^c Overall treatment effect evaluated using the F-test.
^d Comparison of transgenic treatments with control using t-criteria.
^e P-values corrected using the method for estimating the average fraction of false hypothesis deviations (FDR).
^f NA = no statistical analysis was performed as most of the data was <LOQ.
^g Statistical difference is indicated by a P-value <0.05.

Example 6.6.6. Grain Vitamin Analysis

The levels of vitamins A, B1, B2, B5, B6, B12, C, D, E, niacin and folic acid were determined in the samples of control corn, unsprayed corn aad-1, aad-1 + quisalophop, aad-1 + 2.4 -D and aad-1 + both drugs. The total results obtained for all locations are shown in Table 21. Vitamins B12, D, and E could not be quantified using the analysis methods used. All average results obtained for vitamins were similar to the values reported in the literature. The results obtained for vitamins, ranges for which are not indicated in the literature (vitamins B5 and C), were similar to the obtained control values (difference from control <22%). When analyzing at different locations, no significant differences were observed, with the exception of vitamins B1, C, and niacin. Using paired t-criteria, significant differences were observed in relation to vitamin B1 between the control and unsprayed plants aad-1, aad-1 + quisalophop and aad-1 + both drugs, but they were not accompanied by significant overall treatment effects or FDR-adjusted p-values . For vitamin C, a significant overall treatment effect was observed along with significant paired t-criteria and FDR-corrected p-values for aad-1 + quisalophop and aad-1 + 2,4-D. Similarly, with respect to niacin, a significant overall treatment effect was observed along with significant paired t-criteria and FDR-corrected p-values in the case of aad-1 + quisalophop and aad-1 + both. A significant paired t-test was obtained for aad-1 + 2,4-D with respect to niacin, but it was not accompanied by a significant overall treatment effect or FDR-corrected p-value. Since no differences between different locations were observed, and the values were in the ranges known from the literature (if any), the differences were not considered biologically significant.

Table 21 Summary analysis of vitamins in corn grain from all locations Vitamins (mg / kg dry weight) Literary Values ^a The overall effect of processing
(Pr> F) ^b The control Unsprayed (P-value, ^c adjusted P) ^d Sprayed with quisalophop (P-value, adjusted P) Sprinkled 2,4-D (P-value, adjusted P) Sprayed with two drugs (P-value, adjusted P) Beta Carotene (Vitamin A) 0.19–46.8 (0.649) 1.80 1.85
(0.372, 0.566) 1.80
(0.967,
0.983) 1.82
(0.770, 0.883) 1.87
(0.221, 0.376) Vitamin B1 (Thiamine) 1.3-40 (0,068) 3.47 3.63
(0,041 ^e , 0,121) 3.67
(0.013 ^e ,
0,059) 3,54
(0.375, 0.567) 3.64
(0,032 ^e , 0,100) Vitamin B2 (Riboflavin) 0.25-5.6 (0.803) 2.15 2.05
(0.443, 0.631) 2.08
(0.600,
0.756) 1.99
(0.227, 0.383) 2.07
(0.543, 0.708) Vitamin B5 (pantothenic acid) NR ^f (0.820) 5.28 5.17
(0.623, 0.766) 5.09
(0.391,
0.582) 5.29
(0.968, 0.983) 5.10
(0.424, 0.615) Vitamin B6 (Pyridoxine) 3.68–11.3 (0.431) 6.52 6.57
(0.859, 0.938) 6.66
(0.652,
0.782) 6.66
(0.652, 0.782) 7.08
(0,088, 0,210) Vitamin B12 Nr NA ^g <LOQ <LOQ <LOQ <LOQ <LOQ Vitamin C Nr (0.018 ^e ) 22.4 21,2
(0.268, 0.429) 17.5
(0.005 ^e ,
0.028 ^e ) 18.0
(0.004 ^e , 0.026 ^e ) 20,4
(0,068, 0,173) Vitamin D Nr NA <LOQ <LOQ <LOQ <LOQ <LOQ Vitamin E (alpha tocopherol) 1,5-68,7 (0,558) <LOQ <LOQ <LOQ <LOQ <LOQ Niacin (nicotinic acid, vitamin B3)) 9.3-70 (0.013 ^e ) 26.1 24.2
(0,050, 0,140) 22.9
(0.002 ^e ,
0.017 ^e ) 23.7
(0.018 ^e , 0.067) 22.9
(0.002 ^e , 0.016 ^e ) Folic acid 0.15-683 (0.881) 0.594 0.588
(0.779, 0.890) 0.574
(0.403,
0.592) 0.592
(0.931, 0.970) 0.597
(0.916, 0.970) ^a combined range.
^b Overall treatment effect evaluated using the F-test.
^c Comparison of transgenic treatments with control using t-criteria.
^d P-values corrected using the method for estimating the average fraction of false hypothesis deviations (FDR).
^e Statistical difference is indicated by a P-value <0.05.
^f NR = not reported.
^g NA = no statistical analysis was performed as most of the data was <LOQ.

Example 6.6.7. Analysis of anti-nutrients and secondary metabolites in grain

The levels of secondary metabolites (coumaric acid, ferulic acid, furfural and inositol) and anti-nutritional substances (phytic acid, raffinose and trypsin inhibitor) were determined in samples of corn grain in the control, in unsprayed plants aad-1, aad-1 + quisalofop, aad-1 + 2,4-D and aad-1 + both drugs. The total results obtained for all locations are shown in tables 22 and 23. In the case of analysis at different locations, all values were in the ranges known from the literature. No significant differences were observed between the results obtained for aad-1 objects and control objects in the analysis at different locations for inositol and trypsin inhibitor. The results for furfural and raffinose were below the limit of quantitation in this way. Significant paired t-criteria were observed for coumaric acid (unsprayed aad-1, aad-1 + 2,4-D and aad-1 + both drugs) and ferulic acid (aad-1 + quisalofop and aad-1 + both) . Such differences were not accompanied by significant overall treatment effects or FDR-adjusted p-values, and the results were similar to control (difference <10%). Significant overall treatment effect, paired t-test and FDR-corrected p-value were obtained for phytic acid (unsprayed aad-1). Since all the results were in the ranges known from the literature and were similar to the control (difference <11%), such differences were not considered biologically important.

Table 22 The total analysis of secondary metabolites in corn grain from all locations Secondary metabolite (% of dry weight) Literary Values ^a The overall effect of processing
(Pr> F) ^b The control Unsprayed (P-value, ^c adjusted P) ^d Sprayed with quisalophop (P-value, adjusted P) Sprinkled 2,4-D (P-value, adjusted P) Sprayed with two drugs (P-value, adjusted P) Coumaric acid 0.003-0.058 (0,119) 0,021 0,020
(0,038 ^e , 0,113) 0,020
(0,090,
0.211) 0.019
(0,022 ^e , 0,074) 0,020
(0,029 ^e , 0,091) Ferulic acid 0.02-0.389 (0,077) 0.208 0.199
(0.051, 0.141) 0.196
(0.010 ^e ,
0.051) 0,200
(0,080, 0,196) 0.197
(0.019 ^e , 0.069) Furfural 0,0003-0,0006 NA ^f <LOQ <LOQ <LOQ <LOQ <LOQ Inositol 0.0089-0.377 (0.734) 0.218 0.224
(0.548, 0.708) 0.218
(0.973,
0.984) 0.213
(0.612, 0.763) 0.211
(0.526, 0.708) ^a combined range.
^b Overall treatment effect evaluated using the F-test.
^c Comparison of transgenic treatments with control using t-criteria.
^d P-values corrected using the method for estimating the average fraction of false hypothesis deviations (FDR).
^e Statistical difference is indicated by a P-value <0.05.
^f NA = no statistical analysis was performed as most of the data was <LOQ.

Table 23 Summary of anti-nutritional analysis in corn grain from all locations Anti-nutritional substances (% of dry weight) Literary Values ^a The overall effect of processing
(Pr> F) ^b The control Unsprayed (P-value, ^c adjusted P) ^d Sprayed with quisalophop (P-value, adjusted P) Sprinkled 2,4-D (P-value, adjusted P) Sprayed with two drugs (P-value, adjusted P) Phytic acid 0.11-1.57 (0,046 ^e ) 0.727 0.806
(0.003 ^e , 0.020 ^e ) 0.767
(0,099,
0.224) 0.755
(0.245, 0.402) 0.761
(0,158, 0,304) Raffinose 0.02-0.32 NA ^f <LOQ <LOQ <LOQ <LOQ <LOQ Trypsin Inhibitor (TIU / mg) 1.09-7.18 (0.742) 5.08 5.10
(0.954, 0.977) 4.87
(0.631,
0.770) 5.45
(0.387, 0.582) 5.18
(0.813, 0.911) ^a combined range.
^b Overall treatment effect evaluated using the F-test.
^c Comparison of transgenic treatments with control using t-criteria.
^d P-values corrected using the method for estimating the average fraction of false hypothesis deviations (FDR).
^e Statistical difference is indicated by a P-value <0.05.
^f NA = no statistical analysis was performed as most of the data was <LOQ.

Example 7. Additional agronomic tests

The agronomic characteristics of the 40278 maize line were evaluated compared to almost isoline maize under different environmental conditions. The treatment included 4 genetically different hybrids and their corresponding almost isoline control hybrids, which were tested in only 21 locations.

The four tested hybrids were hybrids having medium to late maturity ranging from 99 to 113 days relative maturity. In experiment A, the DAS-40278-9 event was tested in the genetic background in the case of inbred C x BC3S1 conversion. Such a hybrid had a relative maturity of 109 days and was tested at 16 locations (table 24). In experiment B, a hybrid background was tested in the case of conversion of inbred E x BC3S1, a hybrid with a relative maturity of 113 days. Such a hybrid was tested at 14 locations using a group of locations slightly different from the locations in Experiment A (Table 24). In experiments C and D, hybrid backgrounds were tested in the case of BC2S1 x inbred D conversion and BC2S1 x inbred F conversion, respectively. Both of these hybrids have a relative maturity of 99 days, and they were tested in the same 10 locations.

Table 24 Agronomic testing locations
{TC "Table 11. Locations used in agronomic tests in experiment 2" \ f D \ 1 "1"} Location Experiment 2A 2B 2C 2D Atlantic, IA X X Fort Dodge, IA X X X X Huxley, IA X X X X Nora Springs, IA X Wyman, IA X X Lincoln, IL X Pontiac, IL X X X X Princeton, IL X X Seymour, IL X Shannon, IL X X X Viola, IL X X Bremen, IN X X X X Evansville, IN X Fowler, IN X X X X Mt. Vernon, IN X Olivia, MN X X Wayne NE X X York, NE X X Arlington, WI X X X Patteville, WI X X X Watertown, WI X X

For each trial, a randomized full block plan with two repetitions for each location and two row plots was used. The rows were 20 feet (6 m) long and each row was sown with 34 seeds per row. We used standard agronomic practice for the regions during the tests.

Data were collected and analyzed using eight agronomic characteristics: plant height, ear height, stem lodging, root lodging, final population, grain moisture, natural weight and yield. Parameters, plant height and height to the cob, give information about the general form of hybrids. The agronomic characteristics of stem lodging and root lodging in percent determine the possibilities of harvesting the hybrid. Evaluation of the final population allows you to measure seed quality and seasonal growing conditions that affect crop yields. The grain moisture percentage during harvest determines the maturity of the hybrid, and yield (bushels / acre adjusted for moisture) and natural mass (weight in pounds of bushel of corn adjusted for 15.5% moisture) describe the reproductive ability of the hybrid.

Analysis of variance was performed for different field locations using a linear model. The object and location are included in the model as fixed effects. Mixed models were studied, including location and location by objects as random effects, but location by objects explained only a small part of the variance, and the component of the variance often did not significantly differ from zero. In the case of stem and root lodging, a logarithmic transformation was used to stabilize the dispersion, however, the averages and ranges were reported in units of the original scale. Differences were recognized as significant at a 95% confidence level. The significance of the overall treatment effect was evaluated using the t-test.

The results of such tests of agronomic characteristics can be found in table 2. There were no statistically significant differences for any of the four hybrids 40278 compared to the isoline controls (p <0.05) in terms of parameters: height to the cob, stem lodging, root lodging, moisture grain, natural mass and productivity. Estimation of the final population and plant height were statistically different in experiments A and B, respectively, but did not observe similar differences when compared with other tested hybrids 40278. Some observed variability may be due to low levels of genetic variability persisted as a result of backcrossing of plants with the DAS event -40278-9 with elite inbred lines. The total range of measured parameters in all cases was within the values obtained for traditional maize hybrids, and it was impossible to come to the conclusion about increased weed infestation. In conclusion, an analysis of agronomic characteristics shows that 40278 maize is biologically equivalent to ordinary maize.

Table 25 Analysis of agronomic characteristics {TC "Table 12. Analysis of agronomic characteristics from experiment 2" \ f D \ 1 "1"} Experiment A Parameter (units) Treatment Average Range P value Minimum Maximum Plant Height (inches) Aad-1 96.31 94.00 99.00 0.6174 The control 95.41 95.00 98.00 Cob Height (inches) Aad-1 41.08 30.00 48.00 0.4538 The control 44,42 40.00 47.00 Stem lodging (%) Aad-1 3.64 0.00 27.70 0.2020 The control 2.49 0.00 28.57 Root lodging (%) Aad-1 1.00 0.00 7.81 0.7658 The control 0.89 0.00 28.33 Final population (plants / acre per 1000) Aad-1 31.06 27.00 36.00 0,0230 The control 32.17 27.00 36.00 Grain moisture (%) Aad-1 22.10 14.32 27.80 0.5132 The control 21.84 14.52 31.00 Weight (lbs / bushel) Aad-1 54.94 51.10 56.80 0.4123 The control 54.66 51.00 56.80 Productivity (Bushel / Acre) Aad-1 193.50 138.85 229.38 0.9712 The control 187.05 99.87 256.72 Experiment B Parameter (units) Treatment Average Range P value Minimum Maximum Plant Height (inches) Aad-1 106.92 104.00 108.00 0.0178 The control 100.79 95.00 104.00 Cob Height (inches) Aad-1 51.75 49.00 50.00 0.1552 The control 45.63 38.00 50.00 Stem lodging (%) Aad-1 1.24 0.00 15.07 0.1513 The control 0.72 0.00 22.22 Root lodging (%) Aad-1 0.64 0.00 6.15 0.2498 The control 0.40 0.00 9.09 Final population (plants / acre per 1000) Aad-1 31.30 26.00 37.00 0.4001 The control 30.98 25.00 35.00 Grain moisture (%) Aad-1 23.71 14.34 28.70 0.9869 The control 23.72 13.39 31.10 Weight (lbs / bushel) Aad-1 56.96 50.90 59.50 0.2796 The control 56.67 52.00 60.10 Productivity (Bushel / Acre) Aad-1 200.08 102.32 258.36 0.2031 The control 205.41 95.35 259.03 Experiment C Parameter (units) Treatment Average Range P value Minimum Maximum Plant Height (inches) Aad-1 95.92 94.00 96.00 0.1262 The control 90.92 90.00 90.00 Cob Height (inches) Aad-1 47.75 41.00 50.00 0.4630 The control 43.75 37.00 46.00 Stem lodging (%) Aad-1 6.74 0.00 27.47 0.4964 The control 5.46 0.00 28.12 Root lodging (%) Aad-1 0.3512 0.00 7.58 0.8783 The control 0.3077 0.00 33.33 Final population (plants / acre per 1000) Aad-1 32.78 29.00 36.00 0,0543 The control 31.68 24.00 35.00 Grain moisture (%) Aad-1 19.09 13.33 25.90 0.5706 The control 19.36 13.66 26,50 Weight (lbs / bushel) Aad-1 54.62 42.10 58.80 0.1715 The control 55.14 52.80 58.40 Productivity (Bushel / Acre) Aad-1 192.48 135.96 243.89 0.2218 The control 200.35 129.02 285.58 Experiment D Parameter (units) Treatment Average Range P value Minimum Maximum Stem lodging (%) Aad-1 7.29 0.00 9.26 0.4364 The control 4.17 0.00 39.06 Final population (plants / acre per 1000) Aad-1 29.93 27.00 34.00 0,0571 The control 31.86 29.00 35.00 Grain moisture (%) Aad-1 18.74 19.40 24.40 0.4716 The control 19.32 13.35 25.70 Weight (lbs / bushel) Aad-1 56.59 54.80 28.30 0,0992 The control 55.50 52.70 57.40 Productivity (Bushel / Acre) Aad-1 203.55 196.51 240.17 0.7370 The control 199.82 118.56 264.11

Example 8. The use of the insertion site of the event DAS-40278-9 corn for targeted integration

The corresponding agronomic efficacy of the transgene in the case of the DAS-40278-9 event of corn for several generations in the field indicates that the identified areas around the insertion site in the case of the DAS-40278-9 event of the corn provide good positions in the genome for the targeted integration of other transgenic genes of interest . Such targeted integration allows one to overcome the problems associated with the so-called “position effect” and the risk of mutation in the gene during transgene integration in the host. Additional advantages of such targeted integration include, without limitation, the reduction of a large number of transformation events that need to be screened and tested to obtain a transgenic plant that has the desired level of transgene expression, moreover, without manifestation of anomalies as a result of accidental insertion of the transgene into an important locus in the host genome. In addition, such targeted integration allows stacking of transgenes, which makes the elimination of elite plant lines with both genes more efficient.

Using the instructions provided, a person skilled in the art will be able to direct the polynucleic acids of interest to the same insertion site on chromosome 2 as in the case of the corn DAS-40278-9 event, or to the site located immediately near the insertion site in the case of the corn DAS-40278-9 . One such method is disclosed in the international patent application No. WO2008 / 021207, which is incorporated herein by reference in full.

Briefly, up to 20 kb genomic sequence flanking on the 5’ side of the insertion site, and up to 20 kb the genomic sequence flanking on the 3’ side of the insertion site (the parts indicated with reference to the sequence of SEQ ID NO: 29) are used to flank the gene or genes of interest that are supposed to be inserted into the genomic position on chromosome 2 through homologous recombination. The gene or genes of interest can be placed at exactly the same insertion site as in the case of the DAS-40278-9 event of maize, or can be placed elsewhere within areas of 20 kbp. around the insertion sites of the DAS-40278-9 event in maize to provide an appropriate level of transgene expression without adversely affecting the plant. DNA vectors containing a gene or genes of interest and flanking sequences can be delivered to plant cells in one of several ways known to those skilled in the art, including without limitation Agrobacterium mediated transformation. The insertion of donor DNA vectors into the target area of the corn DAS-40278-9 event can be further enhanced in one of several ways, including without limitation co-expression or upregulation of recombination enhancing genes or downregulation of genes that suppress endogenous recombination. In addition, it is known in the art that double-stranded cleavage of specific sequences in the genome can be used to increase the frequency of homologous recombination, thus the insertion into the insertion site of the DAS-40278-9 event in corn and its flanking regions can be enhanced by expression of natural or engineered specific for an endonuclease sequence for cleaving such sequences. Thus, using the instructions provided in the present description, it is possible to embed any heterologous nucleic acid into the chromosome 2 of maize in the target region located between the 5'-molecular marker discussed in Example 4 and the 3'-molecular marker discussed in Example 4 , preferably within the sequence of SEQ ID NO: 29 and / or in its areas, which are discussed in the present description.

Example 9. Excision of the pat gene expression cassette from the DAS-40278-9 event of maize

Removal of a selectable marker gene expression cassette is preferred for targeted insertion of maize DAS-40278-9 into the genomic locus. Removing the selectable pat marker from the DAS-40278-9 event in maize allows reuse of the selectable pat marker in the targeted integration of polynucleic acids into chromosome 2 in subsequent generations of maize.

By applying the described instructions, one skilled in the art is able to excise polynucleic acids of interest from the DAS-40278-9 event of maize. One such method is disclosed in provisional application for the grant of US patent No. 61/297628, which is incorporated into this description by reference in full.

Briefly, sequence-specific endonuclease constructs, such as zinc-finger nucleases, that recognize, bind and cleave specific DNA sequences that flank the gene expression cassette are constructed. Zinc finger nuclease is delivered to the plant cell by crossing the parent plant, which contains the expression cassettes of zinc finger transgenic nucleases, with the second parent plant, in which the maize DAS-40278-9 event takes place. The resulting offspring are grown to maturity and analyzed for the loss of the pat expression cassette by applying to the leaves a herbicide that contains glufosinate. Descendant plants that are not resistant to the herbicide are confirmed at the molecular level and are grown for selfing. Excision and removal of the pat expression cassette is molecularly confirmed in offspring resulting from such self-pollination. Using the instructions provided herein, any heterologous nucleic acid can be excised from corn chromosome 2 in the target region located between the 5'-molecular marker and the 3'-molecular marker, which are discussed in Example 4, preferably within the sequence of SEQ ID NO : 29 or in its designated areas.

Example 10. Resistance to sudden sudden fractures of the stem

A sharp sudden fracture of the stem refers to damage to the corn stalks by strong winds after the application of herbicides-growth regulators, usually during a period of rapid growth. Mechanical impact tests using a rod to physically push the corn by simulating damage by high winds were carried out on hybrid corn in which the DAS-40278-9 event took place, and in control plants in which the DAS-event did not occur 40278-9. The treatments were carried out in four different geographical locations and repeated four times (there was an exception in the case of one test, which was repeated only three times). The plots consisted of eight rows: four rows for each of the two hybrids, with two rows for corn, in which the event DAS-40278-9 takes place, and two rows for corn without an event. Each row was twenty feet (6 m) long. Corn plants were grown prior to developmental stage V4 and a commercial herbicide containing 2,4-D (Weedar 64, Nufarm Inc., Burr Ridge, IL) was added at a rate of 1120 g ec / ha, 2240 g ec / ha and 4480 g ec / ha . Seven days after the application of the herbicide, a mechanical shock test was carried out. A mechanical shock test to investigate sudden sudden fractures consisted in knocking down two rows of corn with a 4-foot (1.2 m) rod to simulate wind damage. The height of the rod and the speed of movement were chosen so as to ensure a low level of stalk breakage (10% or less) in the case of untreated plants to guarantee a test stiff enough to show the difference between treatments. The treatment for sharp fractures was carried out in the opposite direction to the slope of the corn.

In the two locations where the tests were carried out, strong winds and thunderstorms blew 2-3 days after the application of the herbicide 2,4-D. For two consecutive days, a thunderstorm was at Huxley IA. Reported wind speeds from 2 to 17 m sec ^-1 with gusts of 33 m sec ^-1 at the site of the field plot. The direction of the wind was variable. One day a thunderstorm was reported at Lanesboro MN. High winds were reported at the site of the indicated field plot. In addition, it rained during both thunderstorms. The combination of rain and wind explained the sudden sudden fractures reported.

Assessment of sharp fractures in the test with mechanical shock (and severe weather) was carried out thanks to a visual assessment of the percentage of damage. Before machining with a rod causing sharp fractures, we evaluated the density of hybrid corn plants in which the event DAS-40278-9 takes place and controls. A few days after treatment with a rod for abrupt fractures, the density of plants standing on the plot was re-evaluated. Determined the percentage of lodging and the percentage of reduced density of the plant on the plot (table 26). The data obtained in the tests showed that hybrid maize, in which the event DAS-40278-9 took place, is less prone to sudden sudden fractures compared to null plants after the application of 2,4-D.

Table 26 Resistance of maize DAS-40278-9 against sharp fractures to the introduction of 2,4-D-amine in V4. The percentage of abrupt fractures was calculated for hybrid maize plants in which the DAS-40278 event took place, and compared with control plants in which this event did not occur. Before mechanical breaking
The average% lodging 7-8 days after application ¹ Treatment 278 (SLB01-278 //
4XP811XTR) Zero (SLB01 //
4XP811XTR) 278 (SLB01VX-278 //
BE9515XT) Zero (SLB01VX //
BE9515XT) Weedar 64
1120 g ec / ha 0% 38% 0% 33% Weedar 64
2240 g ec / ha one% 42% 0% 33% Weedar 64
4480 g ec / ha 2% 55% one% 46% Unskilled
tanny 0% 0% 0% 0% After mechanical breaking
Average ³ % lodging 11-14 days after application Treatment 278 (SLB01-278 //
4XP811XTR) Zero (SLB01 // 4XP811XTR) 278 (SLB01VX-278 // BE9515XT) Zero (SLB01VX //
BE9515XT) Weedar 64
1120 g ec / ha 0% 19% one% 24% Weedar 64
2240 g ec / ha four% twenty% 7% 27% Weedar 64
4480 g ec / ha four% 26% 6% 28% Unskilled
tanny 0% 0% 0% 0% After mechanical breaking
Average ³ % reduction in standing density 11-14 days after application Treatment 278 (SLB01-278 //
4XP811XTR) Zero (SLB01 // 4XP811XTR) 278 (SLB01VX-278 // BE9515XT) Zero (SLB01VX //
BE9515XT) Weedar 64
1120 g ec / ha 3% 38% 6% 42% Weedar 64
2240 g ec / ha 9% 35% 12% 41% Weedar 64
4480 g ec / ha 9% 40% 16% 40% Unskilled
tanny 0% 0% 0% 0% ¹ Thunderstorm and strong wind 2-3 days after application in two trials.
² Processing repeated four times according to a randomized full block plan (only one test was carried out in three repetitions).
³ Average values adjusted for untreated plants (average for untreated plants was taken as zero values).

Example 11. Analysis of protein in grain

Grain with a high total protein content was obtained from hybrid maize in which the event DAS-40278-9 occurred, compared to control plants that did not have such an event. Two consecutive multicenter field trials were carried out, which included non-sprayed plants and herbicide-treated plants, using combinations of three different herbicides. In 7 out of 8 statistical comparisons, in the case of the DAS-40278-9 event, grains with a significantly higher total protein content were obtained (Table 27). The data obtained were confirmed in the analysis of individual amino acids.

Table 27 Grain Protein Content from Multicenter Field Research Field season 2008 Nontransgenic almost isolinear Event DAS 40278-9, Unsprayed Event DAS 40278-9, Quisalophop,
2,4-D DAS Event 40278-9 Event DAS 40278-9, Quisalophop and 2,4-D Average 9.97 10.9 11.1 10.5 10.9 % increase compared to isoline 0 9.3 11.3 5.3 9.3 Paired t-test Not analyzed 0.002 0,0004 0,061 0.002 Field season 2008 Nontransgenic almost isolinear Event DAS 40278-9, Unsprayed Event DAS 40278-9, Quisalophop,
2,4-D DAS Event 40278-9 Event DAS 40278-9, Quisalophop and 2,4-D Average 10.9 11.6 11.7 11.7 11.5 % increase compared to isoline 0 6.4 7.3 7.2 5.5 Paired t-test Not analyzed 0.0048 0.001 0.0012 0.0079

Example 12. Additional agronomic tests

The agronomic characteristics of hybrid corn, in which the event DAS-40278-9, compared with almost isoline corn, took place, were collected in multicenter field trials, in different geographical conditions during the growing season. Data was collected and analyzed for agronomic characteristics as described in Example 7. The results for hybrid maize lines in which the event DAS-40278-9 occurred compared to null plants are listed in Table 28. In addition, agronomic characteristics for hybrid lines corn, in which the event DAS-40278-9 took place, and null plants sprayed with the herbicide quisalophop (280 g ec / ha) at the developmental stage V3 and sprinkled 2,4-D (2240 g ec / ha) at the developmental stage V6, described in table 29.

Table 28 The results of the determination of yield, moisture percentage and final population for hybrid maize having the event DAS-40278-9, compared with the almost isoline control Title Productivity Grain moisture (%) Final population (plants / acre specified in thousands) Hybrid Corn Containing DAS 40278-9 218.1 21.59 31.69 Hybrid Control Corn 217.4 21.91 30,42

Table 29 Productivity, moisture percentage, root lodging percentage, root lodging percentage, and the general population in the case of hybrid maize lines having the DAS-40278-9 event, compared to the almost isoline control Test Productivity Grain moisture (%) Stem lodging (%) Root lodging (%) Final population (plants /
acre indicated in thousands) Spray Test No. 1 Hybrid Corn Containing DAS 40278-9 214.9 23,4 0.61 2.19 thirty Control hybrid corn No. 1 177.9 23.46 0.97 36.32 28.36 LSD (0.5) 13.3 1,107 0.89 10.7 1,1 No spraying No. 1 Hybrid Corn Containing DAS 40278-9 219.6 22.3 0.95 1.78 30.8 Control hybrid corn No. 1 220.3 22.51 0.54 1,52 30.55 LSD (0.5) 6.9 0,358 0.98 1.65 0.7 Spray Test Hybrid corn No. 2 containing DAS 40278-9 198.6 26.76 0.38 2.08 29.29 Control hybrid corn No. 2 172.3 23.76 1,5 39.16 28.86 LSD (0.5) 13.3 1,107 0.89 10.7 1,1 No spraying Hybrid corn No. 2 containing DAS 40278-9 207.8 24.34 0.22 0.59 31 Control hybrid corn No. 2 206.2 24.88 0.35 0.12 30.94 LSD (0.5) 8.0 0.645 0.55 1.79 0.9

Example 13. Presowing and / or pre-emergence application

Presowing application of herbicides for "burning" weeds are intended for the destruction of weeds that appeared after wintering or in early spring before sowing this crop. Typically, such applications are used in systems where soil preparation by plowing is not used or limited plowing is carried out in which physical removal of weeds does not occur before sowing. Therefore, the herbicidal program must control a very wide range of broad-leaved and cereal weeds present during sowing. Glyphosate, gramoxone and glufosinate are examples of non-selective, non-sequential herbicides widely used for presowing application of "burning" herbicides.

However, it is difficult to control some weeds during this period of the season for one or several of the following reasons: hereditary insensitivity of the species or biotype of the weed to the herbicide, the relatively large size of winter annual weeds and cold weather, which limits the absorption and activity of the herbicides. Several herbicide options are available for preparing tank mixtures with such herbicides in order to expand the weed spectrum and increase weed activity when indiscriminate herbicides are weak. An example would be the use of a 2,4-D tank mixture with glyphosate to control Conyza canadensis (Canadian small-leaved). Glyphosate can be used in a dose of 420 to 1680 g eq / ha, more often from 560 to 840 g eq / ha for pre-sowing type of burning with most available weeds; however, 280-1120 g eq / ha 2,4-D can be used to control many broad-leaved species of weeds (for example, Canadian small-leaved).

2,4-D is the preferred herbicide because it is effective against a very wide range of broadleaf weeds, effective even at low temperatures, and is very inexpensive. However, if the subsequent culture is a sensitive dicotyledonous culture, 2,4-D residues in the soil (albeit short-lived) can adversely affect the culture. Cultures that contain the aad-1 gene are resistant to 2,4-D and are not susceptible to the negative effects of 2,4-D residues in the soil. Increased flexibility and lower cost of introduced components in tank mixtures (or commercial ready-made mixtures) will improve pest control parameters and increase the reliability of burning applications in situations where soil preparation is not applied by plowing or limited plowing is carried out.

Corn aad-1

Transgenic hybrid maize (pDAS 1740-278) containing the aad-1 gene, which encodes an aryloxyalkanoate dioxygenase (AAD-1) protein, was evaluated for resistance to pre-emergence application of 2,4-D in the field. Tests were conducted at several locations in Mississippi, Indiana and Minnesota using a randomized full block plan with three repetitions of two-row plots of approximately 6 m in length at each location. Herbicide-treated plots were paired with untreated plots to provide an accurate assessment of germination and early seasonal growth. Herbicidal treatments of 1120, 2240 and 4480 g ec / ha of 2,4-D-amine were carried out immediately after sowing, but before germination of the culture (0-2 days after sowing). Information on soil and precipitation during such tests is given in table 30.

Table 30 Soil and precipitation information for assessing the resistance of the pDAS1740-278 hybrid to pre-emergence application of the herbicide Test Organic matter,% Texture 1st precipitation (days after application) Rainfall (cm) #one 0.9 Loam one 2.0 # 2 3.3 Silt loam 5 0.3 # 3 1.3 Dusty loam four 1.3

Approximately 16-21 days after sowing and applying 2,4-D, the damage averaged from 12 to 31% in the case of a conventional control hybrid at rates elevated from 1120 to 4480 g ec / ha of 2,4-D-amine. Damage to hybrid maize containing pDAS 1740-278 ranged from 3 to 9% in the same range of application rates. The currently proposed target 2.4-D application rates for transgenic hybrid maize (pDAS 1740-278) containing the aad-1 gene are 1,120 g eq / ha or lower for 2,4-D. Field test results show that hybrid corn containing pDAS 1740-278 has high resistance to 2,4-D herbicide treatments at rates more than two to four times higher than the proposed standards for the intended use, with minimal damage (table 31) .

Table 31 Resistance of pDAS 1740-278 hybrids to pre-emergence application of 2,4-D Herbicide Norm ^a (g ec / ha) Application Stage ^b Damage to plants as a percentage ^c Hybrid pDAS1740-278 Control hybrid 2,4-D amine 1120 Pre 3 12 2,4-D amine 2240 Pre four 16 2,4-D amine 4480 Pre 9 31 ^a ec = equivalent of acid, ha = hectare;
^{b was} applied 0-2 days after sowing before germination;
^c Evaluation 16-21 days after application.

Pre-emergence application of 2,4-D-amine is used at the rates of 1120, 2240, 4480 g eq / ha 7 days, 15 days or 30 days before sowing hybrid corn containing the aad-1 gene and conventional control hybrids. Pre-emergence application is used, using methods known in the art, in field plots that are located in geographically different areas. Herbicide-treated plots are paired with untreated plots to provide an accurate assessment of germination and early seasonal growth. Approximately 16-21 days after sowing and applying 2,4-D 7, 15, or 30 days before sowing, damage to conventional control hybrids and hybrid maize containing aad-1 is measured. Field test results show that hybrid corn containing aad-1 is highly resistant to pre-emergence treatments with 2,4-D herbicide 7, 15, or 30 days before sowing.

Cotton aad-1

Transgenic cotton containing the aad-1 gene, which encodes an aryloxyalkanoate dioxygenase (AAD-1) protein, was evaluated for resistance to pre-emergence application of 2,4-D per field. The trials consisted of three repetitions, and were performed at several locations in Mississippi, Georgia, Tennessee, and Arkansas. A randomized full block plan was used, in which a separate row was separated by a guard row, approximately 10 feet (3 m) long (20 feet (6 m) in the Mississippi trial). Seeds were sown using 8 seeds per foot (0.3 m), then the plants were manually thinned to 3.5 plants / foot in a row. Herbicide-treated plots were paired with untreated plots to provide an accurate assessment of germination and early seasonal growth. All plots received at least ½ inch (12.5 mm) of rain or irrigation water within 24 hours after application. Herbicide treatments of 560, 1120, 2240 and 4480 g ec / ha of 2,4-D-amine (WEEDAR 64, Nufarm, Burr Ridge, II) were carried out immediately after sowing, but before seedling. Information on soil and precipitation during such tests is given in table 32.

Table 32 Soil and precipitation information for the assessment of cotton containing aad-1 Test No. Soil type pH Organic matter,% % sand % sludge % clay #one Silt loam 8.1 1.3 twenty 55 25 # 2 Sandstone # 3 Sandy loam 6.3 0.9 57 34 9 #four Silt loam 6.2 1,1 24 72 four

Pre-emergence application of 2,4-D at a rate of 560, 1120 and 2240 g ec / ha did not significantly affect the standing density of transgenic cotton plants containing the aad-1 gene, compared with untreated plot.

Approximately 7-8 days after sowing and pre-emergence application of 2,4-D at concentrations of 560, 1120, 2240 and 4480 g ec / ha, a decrease in stand density by 3%, no decrease, decrease by 19% and 34%, respectively, were reported , in the case of cotton containing aad-1, compared with a decrease in standing density in the untreated control. 11-14 days after sowing and applying 2,4-D at concentrations of 560, 1120, 2240 and 4480 g eq / ha, a decrease in standing density by 5%, 2%, 14% and 25%, respectively, was reported in the case of cotton containing aad-1, compared with a decrease in standing density in the untreated control.

Pre-emergence application of 2,4-D at a concentration of 560, 1120, 2240 and 4480 g eq / ha did not cause epinastia. Chlorosis was observed at a level of <5% with a visual assessment 7-8 days after application in the case of treatments of 2240 and 4480 g eq / ha. Chlorosis was not detected in the case of treatments of 560 and 1120 g eq / ha on 7-8 days after application. In addition, chlorosis was not detected in cotton containing aad-1 at any of the concentrations of 560, 1120, 2240 and 4480 g ec / ha for the remaining period of the season.

Pre-emergence application of 2,4-D at concentrations of 560 and 1120 g eq / ha resulted in minimal growth inhibition of less than 2% (which is not statistically significant compared to the untreated control plot). The results were constant throughout the test: 7-8, 11-14, 27-30 and 52-57 days after application. Pre-emergence application of 2,4-D at a concentration of 2240 g ec / ha led to growth inhibition of less than 10%. Pre-emergence application of 2,4-D at a concentration of 4480 g eq / ha led to inhibition of growth by 27%, 28%, 17% and 8.3% when evaluated at 7-8, 11-14, 27-30 and 52-57 days after application.

Damage caused by pre-emergence application of 2,4-D at concentrations of 560 and 1120 g ec / ha did not significantly differ from the untreated control plot. Damage estimates for aad-1 cotton plants treated with 2,4-D at a concentration of 2240 g eq / ha ranged from 3% to 15% throughout the test. Damage estimates for aad-1 cotton plants treated with 2,4-D at a concentration of 4480 g eq / ha ranged from 8% to 34% during the entire test.

Differences in plant height, the nature of fruiting and yield were not detected in aad-1 cotton, which was processed using pre-emergence application of 2,4-D at concentrations of 560, 1120, 2240 and 4480 g eq / ha.

Pre-emergence application of 2,4-D-amine was carried out using application rates of 560, 1120, 2240, 4480 g ec / ha, 7 days, 15 days, or 30 days before sowing cotton containing the aad-1 gene and control cotton. Pre-emergence application was carried out using methods known in the art in field plots located at geographically different locations. Herbicide-treated plots are paired with untreated plots to provide an accurate assessment of germination and early seasonal growth. After inoculation and application of 2,4-D 7, 15, or 30 days prior to inoculation, damage to cotton containing the aad-1 gene and control cotton was measured. Field test results show that cotton containing the aad-1 gene is resistant to pre-emergence treatments with 2,4-D herbicide 7, 15, or 30 days before sowing.

This example discusses some of the many options available. Weed control specialists will cite many other applications as examples, including but not limited to gramoxone + 2,4-D or glufosinate + 2,4-D using the products described in the federal herbicide identifier (CPR, 2003) and applications described in the Agriliance Plant Protection Guide (2003). Those skilled in the art will also be aware that the example described above can be applied to any 2,4-D-sensitive (or sensitive to another phenoxy-auxin herbicide) culture that can be protected by the AAD-1 (v3) gene with stable transformation .

Claims (12)

1. A method of weed control, including sowing seeds in a specific area and applying an aryloxyalkanoate herbicide in a specified area 30 days before sowing seeds in a specified area, said seeds containing the AAD-1 aryloxyalkanoate dioxygenase encoded by SEQ ID NO: 29. 2. The method of claim 1, wherein said herbicide is selected from the group consisting of 2,4-D, 2,4-DB, MCPA, MCPB, and aryloxyphenoxy propionate. 3. The method of claim 1, wherein said area is a field and said herbicide is applied within 7 days, 11 days, 14 days, or 15 days before sowing said seeds. 4. The method according to claim 1, used to control glyphosate-resistant weeds in a specified area, while plants from these seeds are grown that additionally have a sign of glyphosate resistance, and the method includes applying an aryloxyalkanoate herbicide to at least a portion of the indicated area. 5. The method according to claim 1, wherein said herbicide is phenoxyiaxin. 6. The method according to p. 4, in which at least one of these weeds is resistant to glyphosate self-seeding of a different species other than the specified plant. 7. The method according to claim 1, wherein said weeds are located on the area to be cultivated, moreover, there are many plants grown from said seeds on said area, said method further comprising applying an aryloxyalkanoate herbicide over the plants. 8. The method according to p. 7, including pre-emergence, post-emergence or pre-emergence and post-emergence application of the herbicide to plants, parts of plants and / or cultivated area. 9. The method according to claim 1, wherein said aryloxyalkanoate herbicide is 2,4-D-amine and said 2,4-D-amine is applied at a rate in the range selected from the group consisting of 560, 1120, 2240 and 4480 g ec / ha. 10. The method according to claim 1, wherein said aryloxyalkanoate herbicide is 2,4-D-amine and said 2,4-D-amine is added at a rate of 280-1120 g eq / ha. 11. The method of claim 1, wherein said seed is obtained from a corn plant. 12. The method of claim 1, wherein said seed contains SEQ ID NO: 29, as is the case in a seed deposited in the American Type Culture Collection (ATCC) with accession number PTA-10244.

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